Benzylisoquinoline alkaloid (BIA) precursor producing microbes, and methods of making and using the same

ABSTRACT

Methods and engineered yeast cells for generating a benzylisoquinoline alkaloid product are provided herein. A method comprises providing engineered yeast cells and a feedstock to a reactor. In the reactor, the engineered yeast cells are subjected to fermentation by incubating the engineered yeast cells for a time period to produce a solution comprising the BIA product and cellular material. The solution comprises not more than one class of molecule selected from the group of protoberberine, morphinan, isopavine, aporphine, and benzylisoquinoline. Additionally, at least one separation unit is used to separate the BIA product from the cellular material to provide the product stream comprising the BIA product.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/567,354 filed Oct. 17, 2017; which applicationis the national phase under 35 U.S.C. 371 of PCT InternationalApplication No. PCT/US2016/030808 filed May 4, 2016 and designating theUnited States, which application, pursuant to 35 U.S.C. § 119(e), claimspriority to the filing date of U.S. Provisional Patent Application Ser.No. 62/156,701 filed on May 4, 2015; the disclosure of which applicationis herein incorporated by reference.

Additionally, this application is related to: U.S. patent applicationSer. No. 14/211,611 now published as US 2014-0273109, which applicationwas filed on Mar. 14, 2014; PCT Application Serial No. PCT/US2014/027833now published as WO2014/143744, which application was filed on Mar. 14,2014; and PCT Application Serial No. PCT/US2014/063738, whichapplication was filed Nov. 3, 2014; the disclosures of whichapplications are herein incorporated by reference.

GOVERNMENT RIGHTS

This invention was made with Government support under contract 1066100awarded by the National Science Foundation and contract DAT007886Aawarded by the National Institutes of Health. The Government has certainrights in the invention.

INTRODUCTION

Benzylisoquinoline alkaloids (BIAs) are a large group of secondarymetabolites from plants and other organisms. These molecules havetherapeutic functions in the human body, ranging from the establishedanalgesic and antitussive properties of morphine and codeine, to novelactivities against cancer and infection observed for molecules such asberberine and sanguinarine. Supply of all these BIA molecules so thatthey are available to researchers and physicians is of interest. Thenumber of synthetic reactions and requirements for selectivestereochemistry means that chemical synthesis of BIAs is low yieldingand not a viable means for large-scale production. Instead, for thewidely used drugs codeine and morphine, the opium poppy (Papaversomniferum) has been bred and developed as a production crop.Intermediates in morphine biosynthesis that find use as drugs and drugprecursors do not accumulate because the plant metabolism is evolved tomaximize pathway flux to the final opioids. Even for end productmetabolites like morphine, accumulation occurs only within specializedcells in the buds and vascular tissue and requires harsh chemicalprocessing of harvested plant material during the extraction process,which may yield less than 2% morphine by dry weight. As such, methodsfor preparing BIAs are of interest.

SUMMARY

Host cells that are engineered to produce benzylisoquinoline alkaloids(BIAs) that are of interest, such as norcoclaurine (NC) andnorlaudanosoline (NL), are provided. BIAs of interest may include BIAprecursors, BIAs, and modifications of BIAs. The host cells may have oneor more modifications selected from: a feedback inhibition alleviatingmutation in an enzyme gene; a transcriptional modulation modification ofa biosynthetic enzyme gene; an inactivating mutation in an enzyme; and aheterologous coding sequence. Also provided are methods of producing aBIA of interest using the host cells and compositions, e.g., kits,systems etc., that find use in methods of the invention.

An aspect of the invention provides a method for forming a productstream having a benzylisoquinoline alkaloid (BIA) product. The methodcomprises providing engineered yeast cells and a feedstock includingnutrients and water to a batch reactor, which engineered yeast cellshave at least one modification selected from the group consisting of: afeedback inhibition alleviating mutation in a biosynthetic enzyme genenative to the cell; a transcriptional modulation modification of abiosynthetic enzyme gene native to the cell; and an inactivatingmutation in an enzyme native to the cell. Additionally, the methodcomprises, in the batch reactor, subjecting the engineered yeast cellsto fermentation by incubating the engineered yeast cells for a timeperiod of at least about 5 minutes to produce a solution comprising theBIA product and cellular material. The method also comprises using atleast one separation unit to separate the BIA product from the cellularmaterial to provide said product stream comprising the BIA product.

In another aspect, the invention provides a method for forming a productstream having a BIA product. The method comprises providing engineeredyeast cells and a feedstock including nutrients and water to a reactor.The method also comprises, in the reactor, subjecting the engineeredyeast cells to fermentation by incubating the engineered yeast cells fora time period of at least about 5 minutes (e.g., 5 minute or longer) toproduce a solution comprising cellular material and the BIA product,wherein the solution comprises not more than one class of moleculeselected from the group of protoberberine, morphinan, isopavine,aporphine and bisbenzylisoquinoline. Additionally, the method comprisesusing at least one separation unit to separate the BIA product from thecellular material to provide the product stream comprising the BIAproduct.

Another aspect of the invention provides an engineered yeast cell thatproduces a benzylisoquinoline alkaloid (BIA) product, the engineeredyeast cell having at least one modification selected from the groupconsisting of: a feedback inhibition alleviating mutation in abiosynthetic enzyme gene native to the cell; a transcriptionalmodulation modification of a biosynthetic enzyme gene native to thecell; and an inactivating mutation in an enzyme native to the cell. Theengineered yeast cell comprises at least one heterologous codingsequence encoding at least one enzyme that is selected from the group of6OMT, CNMT, CYP80B1, CPR and 4′OMT. In some examples, the engineeredyeast cell comprises a plurality of heterologous coding sequencesencoding an enzyme that is selected from the group of 6OMT, CNMT,CYP80B1, CPR and 4′OMT. In some examples, the heterologous codingsequences may be operably connected. Heterologous coding sequences thatare operably connected may be within the same pathway of producing aparticular benzylisoquinoline alkaloid product.

An additional aspect of the invention provides a compound that comprisesa benzylisoquinoline alkaloid product that is characterized as beingpart of at most two classes selected from the group consisting of1-benzylisoquinoline, protoberberine, morphinan, isopavine, aporphine,and bisbenzylisoquinoline. Remaining components of the compound do notcontain a detectable amount of a molecule of a non-selected class fromthe group of 1-benzylisoquinoline, protoberberine, morphinan, isopavine,aporphine and bisbenzylisoquinoline.

In another aspect of the invention, therapeutic agent is provided. Thetherapeutic agent comprises a benzylisoquinoline alkaloid product. Thetherapeutic agent does not contain a detectable amount of an impurityselected from the group consisting of codeine-O(6)-methyl ether,8,14-dihydroxy-7,8-dihydrocodeinone and tetrahydrothebaine.

BRIEF DESCRIPTION OF THE FIGURES

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIG. 1 illustrates the biosynthetic pathway from glucose to tyrosine andother BIA precursor molecules.

FIG. 2 illustrates the effect of ZWF1 knockout and TKL1 over-expressionon the pentose phosphate pathway (PPP). A: native PPP flux, B: ModifiedPPP flux.

FIG. 3 illustrates the synthesis of NC (A) and NL (B) from precursormolecules.

FIG. 4 illustrates the effect of four genetic modifications on NCproduction with varying fed tyrosine.

FIG. 5 shows NC production from strains with combinations of geneticmodifications.

FIG. 6 shows the levels of NL production in aldehyde oxidoreductase(ALD)/alcohol dehydrogenase (ADH) gene knockout strains.

FIG. 7 illustrates the activity of a L-DOPA decarboxylase (DODC) enzymein vivo. Yeast strains transformed with DNA to express Papaversomniferum tyrosine/DOPA decarboxylase may convert L-DOPA to dopamine invivo.

FIG. 8 shows the production of norcoclaurine (NC) in yeast strains fed100 mM dopamine and varying concentrations of tyrosine.

FIG. 9 shows NC production in multiple engineered yeast strains fed 100mM dopamine and no tyrosine.

FIG. 10 shows NC production from dopamine or from L-DOPA in anengineered yeast strain (CSY980) with the additional integration of theL-DOPA decarboxylase PpDODC.

FIG. 11 illustrates a biosynthetic scheme including tyrosinehydroxylation using mammalian tyrosine hydroxylases (TyrHs) with theco-substrate tetrahydrobiopterin (BH4).

FIG. 12 shows that tyrosine hydroxylases expressed from yeast cellsconvert tyrosine to L-DOPA: (A) LC-MS chromatogram confirms conversionof tyrosine to L-DOPA in the presence of co-substrate, BH4; and (B)L-DOPA ion fragmentation in lysate samples.

FIG. 13 shows the co-expression of tyrosine hydroxylase with a BH4biosynthetic enzyme provides for conversion of tyrosine to L-DOPA.

FIG. 14 illustrates the biosynthetic pathways of the BIA precursormolecules through to reticuline going through (A) NC and (B) NL.

FIG. 15 shows LC-MS analysis (A: ion counts) of the production ofNC-derived BIA precursor molecules including N-methylcoclaurine (B: m/zfragmentation pattern) from L-DOPA in the liquid culture of engineeredyeast strains.

FIG. 16 shows LC-MS analysis (ion counts) of the production of (A) NCand (B) reticuline from sugar in the liquid culture of engineered yeaststrains.

FIG. 17 shows the effect of (A) media composition and (B) maltodextrinand amylase concentrations on the production of reticuline from sugar inthe liquid culture of engineered yeast strains.

FIG. 18 shows the effect of inactivating mutations in ADH and ALDenzymes on the production of reticuline from sugar in the liquid cultureof engineering yeast strains.

DEFINITIONS

Before describing exemplary embodiments in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used in the description.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

It is noted that as used herein and in the appended claims, the singularforms “a”, “an”, and “the” include plural referents unless the contextclearly dictates otherwise. For example, the term “a primer” refers toone or more primers, i.e., a single primer and multiple primers. It isfurther noted that the claims are drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation.

As used herein, the terms “determining,” “measuring,” “assessing,” and“assaying” are used interchangeably and include both quantitative andqualitative determinations.

As used herein, the term “polypeptide” refers to a polymeric form ofamino acids of any length, including peptides that range from 2-50 aminoacids in length and polypeptides that are greater than 50 amino acids inlength. The terms “polypeptide” and “protein” are used interchangeablyherein. The term “polypeptide” includes polymers of coded and non-codedamino acids, chemically or biochemically modified or derivatized aminoacids, and polypeptides having modified peptide backbones in which theconventional backbone has been replaced with non-naturally occurring orsynthetic backbones. A polypeptide may be of any convenient length,e.g., 2 or more amino acids, such as 4 or more amino acids, 10 or moreamino acids, 20 or more amino acids, 50 or more amino acids, 100 or moreamino acids, 300 or more amino acids, such as up to 500 or 1000 or moreamino acids. “Peptides” may be 2 or more amino acids, such as 4 or moreamino acids, 10 or more amino acids, 20 or more amino acids, such as upto 50 amino acids. In some embodiments, peptides are between 5 and 30amino acids in length.

As used herein the term “isolated,” refers to an moiety of interest thatis at least 60% free, at least 75% free, at least 90% free, at least 95%free, at least 98% free, and even at least 99% free from othercomponents with which the moiety is associated with prior topurification.

As used herein, the term “encoded by” refers to a nucleic acid sequencewhich codes for a polypeptide sequence, wherein the polypeptide sequenceor a portion thereof contains an amino acid sequence of 3 or more aminoacids, such as 5 or more, 8 or more, 10 or more, 15 or more, or 20 ormore amino acids from a polypeptide encoded by the nucleic acidsequence. Also encompassed by the term are polypeptide sequences thatare immunologically identifiable with a polypeptide encoded by thesequence.

A “vector” is capable of transferring gene sequences to target cells. Asused herein, the terms, “vector construct,” “expression vector,” and“gene transfer vector,” are used interchangeably to mean any nucleicacid construct capable of directing the expression of a gene of interestand which may transfer gene sequences to target cells, which isaccomplished by genomic integration of all or a portion of the vector,or transient or inheritable maintenance of the vector as anextrachromosomal element. Thus, the term includes cloning, andexpression vehicles, as well as integrating vectors.

An “expression cassette” includes any nucleic acid construct capable ofdirecting the expression of a gene/coding sequence of interest, which isoperably linked to a promoter of the expression cassette. Such cassetteis constructed into a “vector,” “vector construct,” “expression vector,”or “gene transfer vector,” in order to transfer the expression cassetteinto target cells. Thus, the term includes cloning and expressionvehicles, as well as viral vectors.

A “plurality” contains at least 2 members. In certain cases, a pluralitymay have 10 or more, such as 100 or more, 1000 or more, 10,000 or more,100,000 or more, 10⁶ or more, 10⁷ or more, 10⁸ or more, or 10⁹ or moremembers.

Numeric ranges are inclusive of the numbers defining the range.

The methods described herein include multiple steps. Each step may beperformed after a predetermined amount of time has elapsed betweensteps, as desired. As such, the time between performing each step may be1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds ormore, 5 minutes or more, 10 minutes or more, 60 minutes or more, andincluding 5 hours or more. In certain embodiments, each subsequent stepis performed immediately after completion of the previous step. In otherembodiments, a step may be performed after an incubation or waiting timeafter completion of the previous step, e.g., a few minutes to anovernight waiting time.

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION

Host cells that are engineered to produce benzylisoquinoline alkaloids(BIAs) that are of interest, such as norcoclaurine (NC) andnorlaudanosoline (NL), are provided. The host cells may have one or moreengineered modifications selected from: a feedback inhibitionalleviating mutation in an enzyme gene; a transcriptional modulationmodification of a biosynthetic enzyme gene; an inactivating mutation inan enzyme; and a heterologous coding sequence. Also provided are methodsof producing a BIA of interest using the host cells and compositions,e.g., kits, systems etc., that find use in methods of the invention.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, and as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein may also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method is carried out in the order of eventsrecited or in any other order which is logically possible.

In further describing the subject invention, BIA precursors of interest,BIAs, and modifications of BIAs are described first in greater detail,followed by host cells for producing the same. Next, methods of interestin which the host cells find use are reviewed. Kits that may be used inpracticing methods of the invention are also described.

Benzylisoouinoline Alkaloid (BIA) Precursors

As summarized above, host cells which produce benzylisoquinolinealkaloid precursors (BIA precursors) are provided. The BIA precursor maybe any intermediate or precursor compound in a synthetic pathway (e.g.,as described herein) that leads to the production of a BIA of interest(e.g., as described herein). In some cases, the BIA precursor has astructure that may be characterized as a BIA or a derivative thereof. Incertain cases, the BIA precursor has a structure that may becharacterized as a fragment of a BIA. In some cases, the BIA precursoris an early BIA. As used herein, by “early BIA” is meant an earlyintermediate in the synthesis of a BIA of interest in a cell, where theearly BIA is produced by a host cell from a host cell feedstock orsimple starting compound. In some cases, the early BIA is a BIAintermediate that is produced by the subject host cell solely from ahost cell feedstock (e.g., a carbon and nutrient source) without theneed for addition of a starting compound to the cells. The term earlyBIA may refer to a precursor of a BIA end product of interest whether ornot the early BIA may itself be characterized as a benzylisoquinolinealkaloid.

In some cases, the BIA precursor is an early BIA, such as apre-reticuline benzylisoquinoline alkaloid. As such, host cells whichproduce pre-reticuline benzylisoquinoline alkaloids (pre-reticulineBIAs) are provided. Reticuline is a major branch point intermediate ofinterest in the synthesis of downstream BIAs via cell engineeringefforts to produce end products such as opioid products. The subjecthost cells may produce BIA precursors from simple and inexpensivestarting materials that may find use in the production of reticuline anddownstream BIA end products.

As used herein, the terms “pre-reticuline benzylisoquinoline alkaloid”,“pre-reticuline BIA”, and “pre-reticuline BIA precursor” are usedinterchangeably and refer to a biosynthetic precursor of reticulinewhether or not the structure of the reticuline precursor itself ischaracterized as a benzylisoquinoline alkaloid. The term pre-reticulineBIA is meant to include biosynthetic precursors, intermediates andmetabolites thereof, of any convenient member of a host cellbiosynthetic pathway that may lead to reticuline. In some cases, thepre-reticuline BIA includes a benzylisoquinoline alkaloid fragment, suchas a benzyl fragment, a quinoline fragment or a precursor or derivativethereof. In certain instances, the pre-reticuline BIA has a structurethat may be characterized as a benzylisoquinoline alkaloid or aderivative thereof.

BIA precursors of interest include, but are not limited to,norcoclaurine (NC) and norlaudanosoline (NL), as well as NC and NLprecursors, such as tyrosine, tyramine, 4-hydroxyphenylacetaldehyde(4-HPA), 4-hydroxyphenylpyruvic acid (4-HPPA),L-3,4-dihydroxyphenylalanine (L-DOPA), 3,4-dihydroxyphenylacetaldehyde(3,4-DHPA), and dopamine. In some embodiments, the one or more BIAprecursors are 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA) and dopamine.In certain instances, the one or more BIA precursors are4-hydroxyphenylacetaldehyde (4-HPA) and dopamine. FIGS. 3A and 3Billustrate the synthesis of NC and NL, respectively from precursormolecules via a Pictet-Spengler condensation reaction, where thereaction may occur spontaneously or may by catalyzed by any convenientenzymes.

Synthetic pathways to a BIA precursor may be generated in the hostcells, and may start with any convenient starting compound(s) ormaterials. FIG. 1 illustrates a synthetic pathway of interest to BIAprecursors starting from glucose. The starting material may benon-naturally occurring or the starting material may be naturallyoccurring in the host cell. Any convenient compounds and materials maybe used as the starting material, based upon the synthetic pathwaypresent in the host cell. The source of the starting material may befrom the host cell itself, e.g., tyrosine, or the starting material maybe added or supplemented to the host cell from an outside source. Assuch, in some cases, the starting compound refers to a compound in asynthetic pathway of the cell that is added to the host cell from anoutside source that is not part of a growth feedstock or cell growthmedia. Starting compounds of interest include, but are not limited to,dopamine, 4-HPA, 4-HPPA, as well as any of the compounds shown inFIG. 1. For example, if the host cells are growing in liquid culture,the cell media may be supplemented with the starting material, which istransported into the cells and converted into the desired products bythe cell. Starting materials of interest include, but are not limitedto, inexpensive feedstocks and simple precursor molecules. In somecases, the host cell utilizes a feedstock including a simple carbonsource as the starting material, which the host cell utilizes to producecompounds of the synthetic pathway of the cell. The host cell growthfeedstock may include one or more components, such as a carbon sourcesuch as cellulose, starch, free sugars and a nitrogen source, such asammonium salts or inexpensive amino acids. In some cases, a growthfeedstock that finds use as a starting material may be derived from asustainable source, such as biomass grown on marginal land, includingswitchgrass and algae, or biomass waste products from other industrialor farming activities.

Benzylisoouinoline Alkaloids (Bias)

As summarized above, host cells which produce benzylisoquinolinealkaloids (BIA) of interest are provided. In some embodiments, theengineered strains of the invention will provide a platform forproducing benzylisoquinoline alkaloids of interest and modificationsthereof across several structural classes including, but not limited to,benzylisoquinolines, protoberberines, protopines, benzophenanthridines,promorphinans, morphinans, secoberberines, phthalideisoquinolines,aporphines, bisbenzylisoquinolines, and others. Each of these classes ismeant to include biosynthetic precursors, intermediates, and metabolitesthereof, of any convenient member of a host cell biosynthetic pathwaythat may lead to a member of the class. Non-limiting examples ofcompounds are given below for each of these structural classes. In someembodiments, the structure of a given example may or may not becharacterized itself as a benzylisoquinoline alkaloid. The presentchemical entities are meant to include all possible isomers, includingsingle enantiomers, racemic mixtures, optically pure forms, mixtures ofdiastereomers and intermediate mixtures.

Benzylisoquinolines may include, but are not limited to, norcoclaurine,norlaudanosoline, coclaurine, 3′-hydroxycoclaurine,4′-O-methylnorlaudanosoline, 4′-O-methyl-laudanosoline,N-methylnorcoclaurine, laudanosoline, N-methylcoclaurine,3′-hydroxy-N-methylcoclaurine, reticuline, norreticuline, papaverine,laudanine, laudanosine, tetrahydropapaverine, 1,2-dihydropapaverine andorientaline.

Protoberberines may include, but are not limited to, scoulerine,cheilanthifoline, stylopine, nandinine, jatrorrhizine, stepholidine,discretamine, cis-N-methylstylopine, tetrahydrocolumbamine, palmatine,tetrahydropalmatine, columbamine, canadine, N-methylcanadine,1-hydroxycanadine, berberine, N-methyl-ophiocarpine,1,13-dihydroxy-N-methylcanadine and1-hydroxy-10-O-acetyl-N-methylcanadine.

Protopines may include, but are not limited to, protopine,6-hydroxyprotopine, allocryptopine, cryptopine, muramine andthalictricine.

Benzophenanthridines may include, but are not limited to,dihydrosanguinarine, sanguinarine, dihydrocheilirubine, cheilirubine,dihydromarcapine, marcapine and chelerythrine.

Promorphinans may include, but are not limited to, salutaridine,salutaridinol and salutaridinol-7-O-acetate.

Morphinans may include, but are not limited to, thebaine, codeinone,codeine, morphine, morphinone, oripavine, neopinone, neopine,neomorphine, hydrocodone, dihydrocodeine, 14-hydroxycodeinone,oxycodone, 14-hydroxycodeine, morphinone, hydromorphone,dihydromorphine, dihydroetorphine, ethylmorphine, etorphine, metopon,buprenorphine, pholcodine, heterocodeine, and oxymorphone.

Secoberberines may include, but are not limited to,4′-O-desmethylmacrantaldehyde, 4′-O-desmethylpapaveroxine,4′-O-desmethyl-3-O-acetylpapaveroxine and 3-O-aceteylpapaveroxine.

Phthalideisoquinolines may include, but are not limited to,narcotolinehemiacetal, narcotinehemiacetal, narcotoline and noscapine.

Aporphines may include, but are not limited to, magnoflorine,corytuberine, apomorphine, boldine, isoboldine, isothebaine,isocorytuberine and glaufine.

Bisbenzylisoquinolines may include, but are not limited to, berbamunine,guattgaumerine, dauricine and liensinine.

Other compounds that may be produced by the engineered strains of theinvention may include, but are not limited to, rhoeadine, pavine,isopavine and cularine.

In certain embodiments, the engineered strains of the invention mayprovide a platform for producing compounds related totetrahydrobiopterin synthesis including, but not limited to,dihydroneopterin triphosphate, 6-pyruvoyl tetrahydropterin,5,6,7,8-tetrahydrobiopterin, 7,8-dihydrobiopterin, tetrahydrobiopterin4a-carbinolamine, quinoid dihydrobiopterin and biopterin.

Host Cells

As summarized above, one aspect of the invention is a host cell thatproduces one or more BIAs of interest. Any convenient cells may beutilized in the subject host cells and methods. In some cases, the hostcells are non-plant cells. In some instances, the host cells may becharacterized as microbial cells. In certain cases, the host cells areinsect cells, mammalian cells, bacterial cells, or yeast cells. Anyconvenient type of host cell may be utilized in producing the subjectBIA-producing cells, see, e.g., US2008/0176754 now published as U.S.Pat. No. 8,975,063, US2014/0273109 and WO2014/143744; the disclosures ofwhich are incorporated by reference in their entirety. Host cells ofinterest include, but are not limited to, bacterial cells, such asBacillus subtilis, Escherichia coli, Streptomyces and Salmonellatyphimuium cells, insect cells such as Drosophila melanogaster S2 andSpodoptera frugiperda Sf9 cells, and yeast cells such as Saccharomycescerevisiae, Schizosaccharomyces pombe, and Pichia pastoris cells. Insome embodiments, the host cells are yeast cells or E. coli cells. Insome cases, the host cell is a yeast cell. In some instances the hostcell is from a strain of yeast engineered to produce a BIA of interest.Any of the host cells described in US2008/0176754 now published as U.S.Pat. No. 8,975,063, US2014/0273109 and WO2014/143744, may be adapted foruse in the subject cells and methods. In certain embodiments, the yeastcells may be of the species Saccharomyces cerevisiae (S. cerevisiae). Incertain embodiments, the yeast cells may be of the speciesSchizosaccharomyces pombe. In certain embodiments, the yeast cells maybe of the species Pichia pastoris. Yeast is of interest as a host cellbecause cytochrome P450 proteins, which are involved in somebiosynthetic pathways of interest, are able to fold properly into theendoplasmic reticulum membrane so that their activity is maintained.Yeast strains of interest that find use in the invention include, butare not limited to, CEN.PK (Genotype: MATa/α ura3-52/ura3-52trp1-289/trp1-289 leu2-3_112/leu2-3_112 his3 Δ1/his3 Δ1 MAL2-8C/MAL2-8CSUC2/SUC2), S288C, W303, D273-10B, X2180, A364A, Σ1278B, AB972, SK1, andFL100. In certain cases, the yeast strain is any of S288C (MATα; SUC2mal mel gal2 CUP1 flo1 flo8-1 hap1), BY4741 (MATα; his3Δ1; leu2Δ0;met15Δ0; ura3Δ0), BY4742 (MATα; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0), BY4743(MATa/MATα; his3Δ1/his3Δ1; leu2Δ0/leu2Δ0; met15Δ0/MET15; LYS2/lys2Δ0;ura3Δ0/ura3Δ0), and WAT11 or W(R), derivatives of the W303-B strain(MATa; ade2-1; his3-11, -15; leu2-3, -112; ura3-1; canR; cyr+) whichexpress the Arabidopsis thaliana NADPH-P450 reductase ATR1 and the yeastNADPH-P450 reductase CPR1, respectively. In another embodiment, theyeast cell is W303alpha (MATα; his3-11, 15 trp1-1 leu2-3 ura3-1 ade2-1).The identity and genotype of additional yeast strains of interest may befound at EUROSCARF(web.uni-frankfurt.de/fb15/mikro/euroscarf/col_index.html).

The host cells may be engineered to include one or more modifications(such as two or more, three or more, four or more, five or more, or evenmore modifications) that provide for the production of BIAs of interest.In some cases, by modification is meant a genetic modification, such asa mutation, addition, or deletion of a gene or fragment thereof, ortranscription regulation of a gene or fragment thereof. In some cases,the one or more (such as two or more, three or more, or four or more)modifications is selected from: a feedback inhibition alleviatingmutation in a biosynthetic enzyme gene native to the cell; atranscriptional modulation modification of a biosynthetic enzyme genenative to the cell; an inactivating mutation in an enzyme native to thecell; and a heterologous coding sequence that encodes an enzyme. A cellthat includes one or more modifications may be referred to as a modifiedcell.

A modified cell may overproduce one or more precursor BIA, BIA, ormodified BIA molecules. By overproduce is meant that the cell has animproved or increased production of a BIA molecule of interest relativeto a control cell (e.g., an unmodified cell). By improved or increasedproduction is meant both the production of some amount of the BIA ofinterest where the control has no BIA precursor production, as well asan increase of about 10% or more, such as about 20% or more, about 30%or more, about 40% or more, about 50% or more, about 60% or more, about80% or more, about 100% or more, such as 2-fold or more, such as 5-foldor more, including 10-fold or more in situations where the control hassome BIA of interest production.

In some cases, the host cell is capable of producing an increased amountof norcoclaurine relative to a control host cell that lacks the one ormore modifications (e.g., as described herein). In certain instances,the increased amount of norcoclaurine is about 10% or more relative tothe control host cell, such as about 20% or more, about 30% or more,about 40% or more, about 50% or more, about 60% or more, about 80% ormore, about 100% or more, 2-fold or more, 5-fold or more, or even10-fold or more relative to the control host cell.

In some cases, the host cell is capable of producing an increased amountof norlaudanosoline relative to a control host cell that lacks the oneor more modifications (e.g., as described herein). In certain instances,the increased amount of norlaudanosoline is about 10% or more relativeto the control host cell, such as about 20% or more, about 30% or more,about 40% or more, about 50% or more, about 60% or more, about 80% ormore, about 100% or more, 2-fold or more, 5-fold or more, or even10-fold or more relative to the control host cell.

In some embodiments, the host cell is capable of producing a 10% or moreyield of norcoclaurine from a starting compound such as tyrosine, suchas 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, or even 90% or more yield of norcoclaurine from astarting compound.

In some embodiments, the host cell is capable of producing a 10% or moreyield of norlaudanosoline from a starting compound such as tyrosine,such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more,70% or more, 80% or more, or even 90% or more yield of norlaudanosolinefrom a starting compound.

In some embodiments, the host cell overproduces one or more BIA ofinterest molecule selected from the group consisting of tyrosine,4-hydroxyphenylacetaldehyde (4-HPA), L-3,4-dihydroxyphenylalanine(L-DOPA), 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA) and dopamine.

Any convenient combinations of the one or more modifications may beincluded in the subject host cells. In some cases, two or more (such astwo or more, three or more, or four or more) different types ofmodifications are included. In certain instances, two or more (such asthree or more, four or more, five or more, or even more) distinctmodifications of the same type of modification are included in thesubject cells.

In some embodiments of the host cell, when the cell includes one or moreheterologous coding sequences that encode one or more enzymes, itincludes at least one additional modification selected from the groupconsisting of: a feedback inhibition alleviating mutations in abiosynthetic enzyme gene native to the cell; a transcriptionalmodulation modification of a biosynthetic enzyme gene native to thecell; and an inactivating mutation in an enzyme native to the cell. Incertain embodiments of the host cell, when the cell includes one or morefeedback inhibition alleviating mutations in one or more biosyntheticenzyme genes native to the cell, it includes a least one additionalmodification selected from the group consisting of: a transcriptionalmodulation modification of a biosynthetic enzyme gene native to thecell; an inactivating mutation in an enzyme native to the cell; and aheterologous coding sequence that encode an enzyme. In some embodimentsof the host cell, when the cell includes one or more transcriptionalmodulation modifications of one or more biosynthetic enzyme genes nativeto the cell, it includes at least one additional modification selectedfrom the group consisting of: a feedback inhibition alleviating mutationin a biosynthetic enzyme gene native to the cell; an inactivatingmutation in an enzyme native to the cell; and a heterologous codingsequence that encodes an enzyme. In certain instances of the host cell,when the cell includes one or more inactivating mutations in one or moreenzymes native to the cell, it includes at least one additionalmodification selected from the group consisting of: a feedbackinhibition alleviating mutation in a biosynthetic enzyme gene native tothe cell; a transcriptional modulation modification of a biosyntheticenzyme gene native to the cell; and a heterologous coding sequence thatencodes an enzyme.

In certain embodiments of the host cell, the cell includes one or morefeedback inhibition alleviating mutations in one or more biosyntheticenzyme genes native to the cell; and one or more transcriptionalmodulation modifications of one or more biosynthetic enzyme gene nativeto the cell. In certain embodiments of the host cell, the cell includesone or more feedback inhibition alleviating mutations in one or morebiosynthetic enzyme genes native to the cell; and one or moreinactivating mutations in an enzyme native to the cell. In certainembodiments of the host cell, the cell includes one or more feedbackinhibition alleviating mutations in one or more biosynthetic enzymegenes native to the cell; and one or more heterologous coding sequences.In some embodiments, the host cell includes one or more modifications(e.g., as described herein) that include one or more of the genes ofinterest described in Table 1.

Feedback Inhibition Alleviating Mutations

In some instances, the host cells are cells that include one or morefeedback inhibition alleviating mutations (such as two or more, three ormore, four or more, five or more, or even more) in one or morebiosynthetic enzyme genes of the cell. In some cases, the one or morebiosynthetic enzyme genes are native to the cell (e.g., is present in anunmodified cell). As used herein, the term “feedback inhibitionalleviating mutation” refers to a mutation that alleviates a feedbackinhibition control mechanism of a host cell. Feedback inhibition is acontrol mechanism of the cell in which an enzyme in the syntheticpathway of a regulated compound is inhibited when that compound hasaccumulated to a certain level, thereby balancing the amount of thecompound in the cell. In some instances, the one or more feedbackinhibition alleviating mutations is in an enzyme described in asynthetic pathway of FIG. 1 or FIG. 2. A mutation that alleviatesfeedback inhibition reduces the inhibition of a regulated enzyme in thecell of interest relative to a control cell and provides for anincreased level of the regulated compound or a downstream biosyntheticproduct thereof. In some cases, by alleviating inhibition of theregulated enzyme is meant that the 10₅₀ of inhibition is increased by2-fold or more, such as by 3-fold or more, 5-fold or more, 10-fold ormore, 30-fold or more, 100-fold or more, 300-fold or more, 1000-fold ormore, or even more. By increased level is meant a level that is 110% ormore of that of the regulated compound in a control cell or a downstreamproduct thereof, such as 120% or more, 130% or more, 140% or more, 150%or more, 160% or more, 170% or more, 180% or more, 190% or more, or 200%or more, such as at least 3-fold or more, at least 5-fold or more, atleast 10-fold or more or even more of the regulated compound in the hostcell or a downstream product thereof.

A variety of feedback inhibition control mechanisms and biosyntheticenzymes native to the host cell that are directed to regulation oflevels of BIA precursors may be targeted for alleviation in the hostcell. The host cell may include one or more feedback inhibitionalleviating mutations in one or more biosynthetic enzyme genes native tothe cell. The mutation may be located in any convenient biosyntheticenzyme genes native to the host cell where the biosynthetic enzyme issubject to regulatory control. In some embodiments, the one or morebiosynthetic enzyme genes encode one or more enzymes selected from a3-deoxy-d-arabinose-heptulosonate-7-phosphate (DAHP) synthase and achorismate mutase. In some embodiments, the one or more biosyntheticenzyme genes encode a 3-deoxy-d-arabinose-heptulosonate-7-phosphate(DAHP) synthase. In some instances, the one or more biosynthetic enzymegenes encode a chorismate mutase. In certain instances, the one or morefeedback inhibition alleviating mutations are present in a biosyntheticenzyme gene selected from ARO4 and ARO7. In certain instances, the oneor more feedback inhibition alleviating mutations are present in abiosynthetic enzyme gene that is ARO4. In certain instances, the one ormore feedback inhibition alleviating mutations are present in abiosynthetic enzyme gene that is ARO7. In some embodiments, the hostcell includes one or more feedback inhibition alleviating mutations inone or more biosynthetic enzyme genes such as one of those genesdescribed in Table 1.

Any convenient numbers and types of mutations may be utilized toalleviate a feedback inhibition control mechanism. As used herein, theterm “mutation” refers to a deletion, insertion, or substitution of anamino acid(s) residue or nucleotide(s) residue relative to a referencesequence or motif. The mutation may be incorporated as a directedmutation to the native gene at the original locus. In some cases, themutation may be incorporated as an additional copy of the geneintroduced as a genetic integration at a separate locus, or as anadditional copy on an episomal vector such as a 2μ or centromericplasmid. In certain instances, the feedback inhibited copy of the enzymeis under the native cell transcriptional regulation. In some instances,feedback inhibited copy of the enzyme is introduced with engineeredconstitutive or dynamic regulation of protein expression by placing itunder the control of a synthetic promoter.

In certain embodiments, the one or more feedback inhibition alleviatingmutations are present in the ARO4 gene. ARO4 mutations of interestinclude, but are not limited to, substitution of the lysine residue atposition 229 with a leucine, a substitution of the glutamine residue atposition 166 with a lysine residue, or a mutation as described byHartmann M, et al. ((2003) Proc. Nat'l Acad. Sci. USA 100(3):862-867) orFukuda et al. ((1992) J Ferment Bioeng 74(2):117-119). In someinstances, mutations for conferring feedback inhibition are selectedfrom a mutagenized library of enzyme mutants. Examples of suchselections include rescue of growth of o-fluoro-D,L-phenylalanine orgrowth of aro3 mutant yeast strains in media with excess tyrosine asdescribed by Fukuda et al. ((1990) Breeding of Brewing Yeast Producing aLarge Amount of Beta-Phenylethyl Alcohol and Beta-Phenylethyl Acetate.Agr Biol Chem Tokyo 54(1):269-271).

ARO7 mutations of interest include, but are not limited to, substitutionof the threonine residue at position 226 with an isoleucine, asdescribed by Schmidheini et al. ((1989), J Bacteriol 171(3):1245-1253)and additional mutations conferring feedback inhibition selected from amutagenized library of microbial chorismate mutase mutants. Examples ofsuch selections include assays for 5-methyltryptophan sensitivity orincreased production of melanin pigments in strains expressingheterologous tyrosinase enzymes (1.9) in the absence of externally fedtyrosine.

In certain embodiments, the host cells of the present invention mayinclude 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 ormore, 13 or more, 14 or more, or even 15 or more feedback inhibitionalleviating mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 feedback inhibition alleviating mutations in one or morebiosynthetic enzyme genes native to the host cell.

Transcriptional Modulation Modifications

The host cells may include one or more transcriptional modulationmodifications (such as two or more, three or more, four or more, five ormore, or even more modifications) of one or more biosynthetic enzymegenes of the cell. In some cases, the one or more biosynthetic enzymegenes are native to the cell. Any convenient biosynthetic enzyme genesof the cell may be targeted for transcription modulation. Bytranscription modulation is meant that the expression of a gene ofinterest in a modified cell is modulated, e.g., increased or decreased,enhanced or repressed, relative to a control cell (e.g., an unmodifiedcell). In some cases, transcriptional modulation of the gene of interestincludes increasing or enhancing expression. By increasing or enhancingexpression is meant that the expression level of the gene of interest isincreased by 2-fold or more, such as by 5-fold or more and sometimes by25-, 50-, or 100-fold or more and in certain embodiments 300-fold ormore or higher, as compared to a control, i.e., expression in the samecell not modified (e.g., by using any convenient gene expression assay).Alternatively, in cases where expression of the gene of interest in acell is so low that it is undetectable, the expression level of the geneof interest is considered to be increased if expression is increased toa level that is easily detectable. In certain instances, transcriptionalmodulation of the gene of interest includes decreasing or repressingexpression. By decreasing or repressing expression is meant that theexpression level of the gene of interest is decreased by 2-fold or more,such as by 5-fold or more and sometimes by 25-, 50-, or 100-fold or moreand in certain embodiments 300-fold or more or higher, as compared to acontrol. In some cases, expression is decreased to a level that isundetectable. Modifications of host cell processes of interest that maybe adapted for use in the subject host cells are described in U.S.Publication No. 20140273109 (Ser. No. 14/211,611) by Smolke et al., thedisclosure of which is herein incorporated by reference in its entirety.

Any convenient biosynthetic enzyme genes may be transcriptionallymodulated, and include but are not limited to, those biosyntheticenzymes described in FIG. 1, such as ARO3, ARO4, ARO1, ARO7, TYR1, TYR,TyrH, DODC, MAO, ARO10, ARO9, and TKL. In some instances, the one ormore biosynthetic enzyme genes is selected from ARO10, ARO9, and TKL. Insome cases, the one or more biosynthetic enzyme genes is ARO10. Incertain instances, the one or more biosynthetic enzyme genes is ARO9. Insome embodiments, the one or more biosynthetic enzyme genes is TKL. Insome embodiments, the host cell includes one or more transcriptionalmodulation modifications to one or more genes such as one of those genesdescribed in Table 1. In some embodiments, the host cell includes one ormore transcriptional modulation modifications to one or more genes suchas one of those genes described in a synthetic pathway of one of FIGS. 1and 2.

In some embodiments, the transcriptional modulation modificationincludes substitution of a strong promoter for a native promoter of theone or more biosynthetic enzyme genes or the expression of an additionalcopy(ies) of the gene or genes under the control of a strong promoter.The promoters driving expression of the genes of interest may beconstitutive promoters or inducible promoters, provided that thepromoters may be active in the host cells. The genes of interest may beexpressed from their native promoters, or non-native promoters may beused. Although not a requirement, such promoters should be medium tohigh strength in the host in which they are used. Promoters may beregulated or constitutive. In some embodiments, promoters that are notglucose repressed, or repressed only mildly by the presence of glucosein the culture medium, are used. There are numerous suitable promoters,examples of which include promoters of glycolytic genes such as thepromoter of the B. subtilis tsr gene (encoding fructose biphosphatealdolase) or GAPDH promoter from yeast S. cerevisiae (coding forglyceraldehyde-phosphate dehydrogenase) (Bitter G. A., Meth. Enzymol.152:673 684 (1987)). Other strong promoters of interest include, but arenot limited to, the ADHI promoter of baker's yeast (Ruohonen L., et al,J. Biotechnol. 39:193 203 (1995)), the phosphate-starvation inducedpromoters such as the PHOS promoter of yeast (Hinnen, A., et al, inYeast Genetic Engineering, Barr, P. J., et al. eds, Butterworths (1989),the alkaline phosphatase promoter from B. licheniformis (Lee. J. W. K.,et al., J. Gen. Microbiol. 137:1127 1133 (1991)), GPD1 and TEF1. Yeastpromoters of interest include, but are not limited to, induciblepromoters such as Gal1-10, Gal1, GalL, GalS, repressible promoter Met25,tetO, and constitutive promoters such as glyceraldehyde 3-phosphatedehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH),translation-elongation factor-1-alpha promoter (TEF), cytochromec-oxidase promoter (CYC1), MRP7 promoter, etc. In some instances, thestrong promoter is GPD1. In certain instances, the strong promoter isTEF1. Autonomously replicating yeast expression vectors containingpromoters inducible by hormones such as glucocorticoids, steroids, andthyroid hormones are also known and include, but are not limited to, theglucorticoid responsive element (GRE) and thyroid hormone responsiveelement (TRE), see e.g., those promoters described in U.S. Pat. No.7,045,290. Vectors containing constitutive or inducible promoters suchas alpha factor, alcohol oxidase, and PGH may be used. Additionally anypromoter/enhancer combination (as per the Eukaryotic Promoter Data BaseEPDB) could also be used to drive expression of genes of interest. It isunderstood that any convenient promoters specific to the host cell maybe selected, e.g., E. coli. In some cases, promoter selection may beused to optimize transcription, and hence, enzyme levels to maximizeproduction while minimizing energy resources.

Inactivating Mutations

The host cells may include one or more inactivating mutations to anenzyme of the cell (such as two or more, three or more, four or more,five or more, or even more). The inclusion of one or more inactivatingmutations may modify the flux of a synthetic pathway of a host cell toincrease the levels of a BIA of interest or a desirable enzyme orprecursor leading to the same. In some cases, the one or moreinactivating mutations are to an enzyme native to the cell. FIG. 2illustrates a native pentose phosphate pathway (PPP) flux and modifiedPPP flux where that involves inactivation of ZWF1 enzyme. As usedherein, by “inactivating mutation” is meant one or more mutations to agene or regulatory DNA sequence of the cell, where the mutation(s)inactivates a biological activity of the protein expressed by that geneof interest. In some cases, the gene is native to the cell. In someinstances, the gene encodes an enzyme that is inactivated and is part ofor connected to the synthetic pathway of a BIA of interest produced bythe host cell. In some instances, an inactivating mutation is located ina regulatory DNA sequence that controls a gene of interest. In certaincases, the inactivating mutation is to a promoter of a gene. Anyconvenient mutations (e.g., as described herein) may be utilized toinactivate a gene or regulatory DNA sequence of interest. By“inactivated” or “inactivates” is meant that a biological activity ofthe protein expressed by the mutated gene is reduced by 10% or more,such as by 20% or more, 30% or more, 40% or more, 50% or more, 60% ormore, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more,or 99% or more, relative to a control protein expressed by a non-mutatedcontrol gene. In some cases, the protein is an enzyme and theinactivating mutation reduces the activity of the enzyme.

In some embodiments, the cell includes an inactivating mutation in anenzyme native to the cell. Any convenient enzymes may be targeted forinactivation. Enzymes of interest include, but are not limited to thoseenzymes, described in FIGS. 1 and 2 whose action in the syntheticpathway of the host cell tends to reduce the levels of a BIA ofinterest. In some cases, the enzyme has glucose-6-phosphatedehydrogenase activity. In certain embodiments, the enzyme that includesan inactivating mutation is ZWF1 (see e.g., FIG. 2). In some cases, theenzyme has alcohol dehydrogenase activity. In some embodiments, theenzyme that includes an inactivating mutation is selected from ADH2,ADH3, ADH4, ADH5, ADH6, ADH7, and SFA1. In certain embodiments, theenzyme that includes an inactivating mutation(s) is ADH2. In certainembodiments, the enzyme that includes an inactivating mutation(s) isADH3. In certain embodiments, the enzyme that includes an inactivatingmutation(s) is ADH4. In certain embodiments, the enzyme that includes aninactivating mutation(s) is ADH5. In certain embodiments, the enzymethat includes an inactivating mutation(s) is ADH6. In certainembodiments, the enzyme that includes an inactivating mutation(s) isADH7. In some cases, the enzyme has aldehyde oxidoreductase activity. Incertain embodiments, the enzyme that includes an inactivating mutationis selected from ALD2, ALD3, ALD4, ALD5, and ALD6. In certainembodiments, the enzyme that includes an inactivating mutation(s) isALD2. In certain embodiments, the enzyme that includes an inactivatingmutation(s) is ALD3. In certain embodiments, the enzyme that includes aninactivating mutation(s) is ALD4. In certain embodiments, the enzymethat includes an inactivating mutation(s) is ALD5. In certainembodiments, the enzyme that includes an inactivating mutation(s) isALD6. In some embodiments, the host cell includes one or moreinactivating mutations to one or more genes described in Table 1.

Heterologous Coding Sequences

In some instances, the host cells are cells that harbor one or moreheterologous coding sequences (such as two or more, three or more, fouror more, five or more, or even more) which encode activity(ies) thatenable the host cells to produce desired BIAs of interest, e.g., asdescribed herein. As used herein, the term “heterologous codingsequence” is used to indicate any polynucleotide that codes for, orultimately codes for, a peptide or protein or its equivalent amino acidsequence, e.g., an enzyme, that is not normally present in the hostorganism and may be expressed in the host cell under proper conditions.As such, “heterologous coding sequences” includes multiple copies ofcoding sequences that are normally present in the host cell, such thatthe cell is expressing additional copies of a coding sequence that arenot normally present in the cells. The heterologous coding sequences maybe RNA or any type thereof, e.g., mRNA, DNA or any type thereof, e.g.,cDNA, or a hybrid of RNA/DNA. Coding sequences of interest include, butare not limited to, full-length transcription units that include suchfeatures as the coding sequence, introns, promoter regions, 3′-UTRs, andenhancer regions.

In examples, the engineered host cell comprises a plurality ofheterologous coding sequences each encoding an enzyme. In some examples,the plurality of enzymes encoded by the plurality of heterologous codingsequences may be distinct from each other. In some examples, some theplurality of enzymes encoded by the plurality of heterologous codingsequences may be distinct from each other and some of the plurality ofenzymes encoded by the plurality of heterologous coding sequences may beduplicate copies.

In some examples, the heterologous coding sequences may be operablyconnected. Heterologous coding sequences that are operably connected maybe within the same pathway of producing a particular benzylisoquinolinealkaloid product. In some examples, the operably connected heterologouscoding sequences may be directly sequential along the pathway ofproducing a particular benzylisoquinoline alkaloid product. In someexamples, the operably connected heterologous coding sequences may haveone or more native enzymes between one or more of the enzymes encoded bythe plurality of heterologous coding sequences. In some examples, theheterologous coding sequences may have one or more heterologous enzymesbetween one or more of the enzymes encoded by the plurality ofheterologous coding sequences. In some examples, the heterologous codingsequences may have one or more non-native enzymes between one or more ofthe enzymes encoded by the plurality of heterologous coding sequences.

In some embodiments, the host cell includes norcoclaurine (NC) synthaseactivity. Any convenient NC synthase enzymes find use in the subjecthost cells. NC synthase enzymes of interest include, but are not limitedto, enzymes such as EC 4.2.1.78, as described in Table 1. In certainembodiments, the host cell includes a heterologous coding sequence foran NC synthase or an active fragment thereof. In some instances, thehost cell includes one or more heterologous coding sequences for one ormore enzymes or active fragments thereof that convert tyrosine toL-DOPA. In certain cases, the one or more enzymes is selected frombacterial tyrosinases, eukaryotic tyrosinases (e.g., EC 1.14.18.1) andtyrosine hydroxylases (e.g., EC 1.14.16.2.) In some instances, the hostcell includes one or more heterologous coding sequences for one or moreenzymes or active fragments thereof that convert L-DOPA to dopamine(e.g., EC 4.1.1.28).

In certain embodiments, the cell includes one or more heterologouscoding sequences for one or more enzymes or active fragments thereofthat convert dopamine to 3,4-DHPA. In certain cases, the one or moreenzymes is a monoamine oxidase (MAO) (e.g., EC 1.4.3.4). The one or moreheterologous coding sequences may be derived from any convenient species(e.g., as described herein). In some cases, the one or more heterologouscoding sequences may be derived from a species described in Table 1. Insome cases, the one or more heterologous coding sequences are present ina gene or enzyme selected from those described in Table 1.

In some instances, the one or more heterologous coding sequences includea MAO coding sequence integrated at a genomic locus encoding nativeARO10. In certain instances, the one or more heterologous codingsequences include a MAO coding sequence operably linked to an induciblepromoter. In some embodiments, the inducible promoter is part of aninducible system including a DNA binding protein targeted to a promoterregulating the ARO10 gene. In some embodiments, the host cell includesone or more heterologous coding sequences for one or more enzymes oractive fragments thereof described in the genes of Table 1.

As used herein, the term “heterologous coding sequences” also includesthe coding portion of the peptide or enzyme, i.e., the cDNA or mRNAsequence, of the peptide or enzyme, as well as the coding portion of thefull-length transcriptional unit, i.e., the gene including introns andexons, as well as “codon optimized” sequences, truncated sequences orother forms of altered sequences that code for the enzyme or code forits equivalent amino acid sequence, provided that the equivalent aminoacid sequence produces a functional protein. Such equivalent amino acidsequences may have a deletion of one or more amino acids, with thedeletion being N-terminal, C-terminal, or internal. Truncated forms areenvisioned as long as they have the catalytic capability indicatedherein. Fusions of two or more enzymes are also envisioned to facilitatethe transfer of metabolites in the pathway, provided that catalyticactivities are maintained.

Operable fragments, mutants or truncated forms may be identified bymodeling and/or screening. This is made possible by deletion of, forexample, N-terminal, C-terminal, or internal regions of the protein in astep-wise fashion, followed by analysis of the resulting derivative withregard to its activity for the desired reaction compared to the originalsequence. If the derivative in question operates in this capacity, it isconsidered to constitute an equivalent derivative of the enzyme proper.

Aspects of the present invention also relate to heterologous codingsequences that code for amino acid sequences that are equivalent to thenative amino acid sequences for the various enzymes. An amino acidsequence that is “equivalent” is defined as an amino acid sequence thatis not identical to the specific amino acid sequence, but rathercontains at least some amino acid changes (deletions, substitutions,inversions, insertions, etc.) that do not essentially affect thebiological activity of the protein as compared to a similar activity ofthe specific amino acid sequence, when used for a desired purpose. Thebiological activity refers to, in the example of a decarboxylase, itscatalytic activity. Equivalent sequences are also meant to include thosewhich have been engineered and/or evolved to have properties differentfrom the original amino acid sequence. Mutable properties of interestinclude catalytic activity, substrate specificity, selectivity,stability, solubility, localization, etc. In certain embodiments, an“equivalent” amino acid sequence contains at least 80%-99% identity atthe amino acid level to the specific amino acid sequence, in some casesat least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and morein certain cases, at least 95%, 96%, 97%, 98% and 99% identity, at theamino acid level. In some cases, the amino acid sequence may beidentical but the DNA sequence is altered such as to optimize codonusage for the host organism, for example.

The host cells may also be modified to possess one or more geneticalterations to accommodate the heterologous coding sequences.Alterations of the native host genome include, but are not limited to,modifying the genome to reduce or ablate expression of a specificprotein that may interfere with the desired pathway. The presence ofsuch native proteins may rapidly convert one of the intermediates orfinal products of the pathway into a metabolite or other compound thatis not usable in the desired pathway. Thus, if the activity of thenative enzyme were reduced or altogether absent, the producedintermediates would be more readily available for incorporation into thedesired product.

In some instances, where ablation of expression of a protein may be ofinterest, the alteration is in proteins involved in the pleiotropic drugresponse, including, but not limited to, ATP-binding cassette (ABC)transporters, multidrug resistance (MDR) pumps, and associatedtranscription factors. These proteins are involved in the export of BIAmolecules into the culture medium, thus deletion controls the export ofthe compounds into the media, making them more available forincorporation into the desired product. In some embodiments, host cellgene deletions of interest include genes associated with the unfoldedprotein response and endoplasmic reticulum (ER) proliferation. Such genedeletions may lead to improved BIA production. The expression ofcytochrome P450s may induce the unfolded protein response and may causethe ER to proliferate. Deletion of genes associated with these stressresponses may control or reduce overall burden on the host cell andimprove pathway performance. Genetic alterations may also includemodifying the promoters of endogenous genes to increase expressionand/or introducing additional copies of endogenous genes. Examples ofthis include the construction/use of strains which overexpress theendogenous yeast NADPH-P450 reductase CPR1 to increase activity ofheterologous P450 enzymes. In addition, endogenous enzymes such as ARO8,9, and 10, which are directly involved in the synthesis of intermediatemetabolites, may also be overexpressed.

Heterologous coding sequences of interest include but are not limited tosequences that encode enzymes, either wild-type or equivalent sequences,that are normally responsible for the production of BIAs and precursorsin plants. In some cases, the enzymes for which the heterologoussequences code may be any of the enzymes in the BIA pathway, and may befrom any convenient source. The choice and number of enzymes encoded bythe heterologous coding sequences for the particular synthetic pathwaymay be selected based upon the desired product. In certain embodiments,the host cells of the present invention may include 1 or more, 2 ormore, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more,9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more,or even 15 or more heterologous coding sequences, such as 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 heterologous coding sequences.

In some cases, polypeptide sequences encoded by the heterologous codingsequences are as reported in GENBANK. Enzymes of interest include, butare not limited to, those enzymes described herein and those shown inTable 1. The host cells may include any combination of the listedenzymes, from any source. Unless otherwise indicated, accession numbersin Table 1 refer to GenBank. Some accession numbers refer to theSaccharomyces genome database (SGD), which is available on theworld-wide web at www.yeastgenome.org.

In some embodiments, the host cell (e.g., a yeast strain) is engineeredfor selective production of a BIA of interest by localizing one or moreenzymes to a compartment in the cell. In some cases, an enzyme may belocated in the host cell such that the compound produced by this enzymespontaneously rearranges, or is converted by another enzyme to adesirable metabolite before reaching a localized enzyme that may convertthe compound into an undesirable metabolite. The spatial distancebetween two enzymes may be selected to prevent one of the enzymes fromacting directly on a compound to make an undesirable metabolite, andrestrict production of undesirable end products (e.g., an undesirableopioid by-product). In certain embodiments, any of the enzymes describedherein, either singularly or together with a second enzyme, may belocalized to any convenient compartment in the host cell, including butnot limited to, an organelle, endoplasmic reticulum, golgi, vacuole,nucleus, plasma membrane, or the periplasm. In some embodiments, thehost cell includes one or more of the enzymes that include alocalization tag. Any convenient tags may be utilized. In some cases,the localization tag is a peptidic sequence that is attached at theN-terminal and/or C-terminal of the enzyme.

Any convenient methods may be utilized for attaching a tag to theenzyme. In some cases, the localization tag is derived from anendogenous yeast protein. Such tags may provide a route to a variety ofyeast organelles including, but not limited to, the endoplasmicreticulum (ER), mitochondria (MT), plasma membrane (PM), and vacuole(V). In certain embodiments, the tag is an ER routing tag (e.g., ER1).In certain embodiments, the tag is a vacuole tag (e.g., V1). In certainembodiments, the tag is a plasma membrane tag (e.g., P1). In certaininstances, the tag includes or is derived from, a transmembrane domainfrom within the tail-anchored class of proteins. In some embodiments,the localization tag locates the enzyme on the outside of an organelle.In certain embodiments, the localization tag locates the enzyme on theinside of an organelle.

In some instances, the expression of each type of enzyme is increasedthrough additional gene copies (i.e., multiple copies), which increasesintermediate accumulation and/or BIA of interest production. Embodimentsof the present invention include increased BIA of interest production ina host cell through simultaneous expression of multiple species variantsof a single or multiple enzymes. In some cases, additional gene copiesof a single or multiple enzymes are included in the host cell. Anyconvenient methods may be utilized including multiple copies of aheterologous coding sequence for an enzyme in the host cell.

In some embodiments, the host cell includes multiple copies of aheterologous coding sequence for an enzyme, such as 2 or more, 3 ormore, 4 or more, 5 or more, or even 10 or more copies. In certainembodiments, the host cell includes multiple copies of heterologouscoding sequences for one or more enzymes, such as multiple copies of twoor more, three or more, four or more, etc. In some cases, the multiplecopies of the heterologous coding sequence for an enzyme are derivedfrom two or more different source organisms as compared to the hostcell. For example, the host cell may include multiple copies of oneheterologous coding sequence, where each of the copies is derived from adifferent source organism. As such, each copy may include somevariations in explicit sequences based on inter-species differences ofthe enzyme of interest that is encoded by the heterologous codingsequence.

In some embodiments of the host cell, the heterologous coding sequenceis from a source organism selected from the group consisting of P.somniferum, T. flavum, and C. japonica. In some instances, the sourceorganism is P. somniferum, E. californica, C. japonica, T. flavum,Berberis stolonifer, T. flavum subsp. glaucum, Coptis chinensis,Thalictrum spp, Coptis spp, Papaver spp, Berberis wilsonae, A. mexicana,or Berberis spp. In certain instances, the heterologous coding sequenceis from a source organism selected from P. somniferum, T. flavum, and C.japonica. In some embodiments, the host cell includes a heterologouscoding sequence from one or more of the source organisms described inTable 1.

The engineered host cell medium may be sampled and monitored for theproduction of BIAs of interest. The BIAs of interest may be observed andmeasured using any convenient methods. Methods of interest include, butare not limited to, LC-MS methods (e.g., as described herein) where asample of interest is analyzed by comparison with a known amount of astandard compound. Identity may be confirmed, e.g., by m/z and MS/MSfragmentation patterns, and quantitation or measurement of the compoundmay be achieved via LC trace peaks of know retention time and/or EIC MSpeak analysis by reference to corresponding LC-MS analysis of a knownamount of a standard of the compound.

Methods

Process Steps

As summarized above, aspects of the invention include methods ofpreparing a benzylisoquinoline alkaloid (BIA) of interest. As such,aspects of the invention include culturing a host cell under conditionsin which the one or more host cell modifications (e.g., as describedherein) are functionally expressed such that the cell converts startingcompounds of interest into product BIAs of interest or precursorsthereof (e.g., pre-reticuline BIAs). Also provided are methods thatinclude culturing a host cell under conditions suitable for proteinproduction such that one or more heterologous coding sequences arefunctionally expressed and convert starting compounds of interest intoproduct BIAs of interest. In some instances, the method is a method ofpreparing a benzylisoquinoline alkaloid (BIA), include culturing a hostcell (e.g., as described herein); adding a starting compound to the cellculture; and recovering the BIA from the cell culture. In someembodiments of the method, the starting compound, BIA product and hostcell are described by one of the entries of Table 1.

Fermentation media may contain suitable carbon substrates. The source ofcarbon suitable to perform the methods of this disclosure may encompassa wide variety of carbon containing substrates. Suitable substrates mayinclude, without limitation, monosaccharides (e.g., glucose, fructose,galactose, xylose), oligosaccharides (e.g., lactose, sucrose,raffinose), polysaccharides (e.g., starch, cellulose), or a combinationthereof. In some cases, unpurified mixtures from renewable feedstocksmay be used (e.g., cornsteep liquor, sugar beet molasses, barley malt).In some cases, the carbon substrate may be a one-carbon substrate (e.g.,methanol, carbon dioxide) or a two-carbon substrate (e.g., ethanol). Inother cases, other carbon containing compounds may be utilized, forexample, methylamine, glucosamine, and amino acids.

Any convenient methods of culturing host cells may be employed forproducing the BIA precursors and downstream BIAs of interest. Theparticular protocol that is employed may vary, e.g., depending on hostcell, the heterologous coding sequences, the desired BIA precursors anddownstream BIAs of interest, etc. The cells may be present in anyconvenient environment, such as an environment in which the cells arecapable of expressing one or more functional heterologous enzymes. Invitro, as used herein, simply means outside of a living cell, regardlessof the location of the cell. As used herein, the term in vivo indicatesinside a living cell, regardless of the location of the cell. In someembodiments, the cells are cultured under conditions that are conduciveto enzyme expression and with appropriate substrates available to allowproduction of BIA precursors and downstream BIAs of interest in vivo. Insome embodiments, the functional enzymes are extracted from the host forproduction of BIAs under in vitro conditions. In some instances, thehost cells are placed back into a multicellular host organism. The hostcells are in any phase of growth, including, but not limited to,stationary phase and log-growth phase, etc. In addition, the culturesthemselves may be continuous cultures or they may be batch cultures.

Cells may be grown in an appropriate fermentation medium at atemperature between 20-40° C. Cells may be grown with shaking at anyconvenient speed (e.g., 200 rpm). Cells may be grown at a suitable pH.Suitable pH ranges for the fermentation may be between pH 5-9.Fermentations may be performed under aerobic, anaerobic, or microaerobicconditions. Any suitable growth medium may be used. Suitable growthmedia may include, without limitation, common commercially preparedmedia such as synthetic defined (SD) minimal media or yeast extractpeptone dextrose (YEPD) rich media. Any other rich, defined, orsynthetic growth media appropriate to the microorganism may be used.

Cells may be cultured in a vessel of essentially any size and shape.Examples of vessels suitable to perform the methods of this disclosuremay include, without limitation, multi-well shake plates, test tubes,flasks (baffled and non-baffled), and bioreactors. The volume of theculture may range from 10 microliters to greater than 10,000 liters.

The addition of agents to the growth media that are known to modulatemetabolism in a manner desirable for the production of alkaloids may beincluded. In a non-limiting example, cyclic adenosine 2′3′-monophosphatemay be added to the growth media to modulate catabolite repression.

Any convenient cell culture conditions for a particular cell type may beutilized. In certain embodiments, the host cells that include one ormore modifications are cultured under standard or readily optimizedconditions, with standard cell culture media and supplements. As oneexample, standard growth media when selective pressure for plasmidmaintenance is not required may contain 20 g/L yeast extract, 10 g/Lpeptone, and 20 g/L dextrose (YPD). Host cells containing plasmids aregrown in synthetic complete (SC) media containing 1.7 g/L yeast nitrogenbase, 5 g/L ammonium sulfate, and 20 g/L dextrose supplemented with theappropriate amino acids required for growth and selection. Alternativecarbon sources which may be useful for inducible enzyme expressioninclude, but are not limited to, sucrose, raffinose, and galactose.Cells are grown at any convenient temperature (e.g., 30° C.) withshaking at any convenient rate (e.g., 200 rpm) in a vessel, e.g., intest tubes or flasks in volumes ranging from 1-1000 mL, or larger, inthe laboratory.

Culture volumes may be scaled up for growth in larger fermentationvessels, for example, as part of an industrial process. The industrialfermentation process may be carried out under closed-batch, fed-batch,or continuous chemostat conditions, or any suitable mode offermentation. In some cases, the cells may be immobilized on a substrateas whole cell catalysts and subjected to fermentation conditions foralkaloid production.

A batch fermentation is a closed system, in which the composition of themedium is set at the beginning of the fermentation and not alteredduring the fermentation process. The desired organism(s) are inoculatedinto the medium at the beginning of the fermentation. In some instances,the batch fermentation is run with alterations made to the system tocontrol factors such as pH and oxygen concentration (but not carbon). Inthis type of fermentation system, the biomass and metabolitecompositions of the system change continuously over the course of thefermentation. Cells typically proceed through a lag phase, then to a logphase (high growth rate), then to a stationary phase (growth ratereduced or halted), and eventually to a death phase (if left untreated).

A fed-batch fermentation is similar to a batch fermentation, except thatthe substrate is added in intervals to the system over the course of thefermentation process. Fed-batch systems are used to reduce the impact ofcatabolite repression on the metabolism of the host cells and underother circumstances where it is desired to have limited amounts ofsubstrate in the growth media.

A continuous fermentation is an open system, in which a definedfermentation medium is added continuously to the bioreactor and an equalamount of fermentation media is continuously removed from the vessel forprocessing. Continuous fermentation systems are generally operated tomaintain steady state growth conditions, such that cell loss due tomedium being removed must be balanced by the growth rate in thefermentation. Continuous fermentations are generally operated atconditions where cells are at a constant high cell density. Continuousfermentations allow for the modulation of one or more factors thataffect target product concentration and/or cell growth.

The liquid medium may include, but is not limited to, a rich orsynthetic defined medium having an additive component described above.Media components may be dissolved in water and sterilized by heat,pressure, filtration, radiation, chemicals, or any combination thereof.Several media components may be prepared separately and sterilized, andthen combined in the fermentation vessel. The culture medium may bebuffered to aid in maintaining a constant pH throughout thefermentation.

Process parameters including temperature, dissolved oxygen, pH,stirring, aeration rate, and cell density may be monitored or controlledover the course of the fermentation. For example, temperature of afermentation process may be monitored by a temperature probe immersed inthe culture medium. The culture temperature may be controlled at the setpoint by regulating the jacket temperature. Water may be cooled in anexternal chiller and then flowed into the bioreactor control tower andcirculated to the jacket at the temperature required to maintain the setpoint temperature in the vessel.

Additionally, a gas flow parameter may be monitored in a fermentationprocess. For example, gases may be flowed into the medium through asparger. Gases suitable for the methods of this disclosure may includecompressed air, oxygen, and nitrogen. Gas flow may be at a fixed rate orregulated to maintain a dissolved oxygen set point.

The pH of a culture medium may also be monitored. In examples, the pHmay be monitored by a pH probe that is immersed in the culture mediuminside the vessel. If pH control is in effect, the pH may be adjusted byacid and base pumps which add each solution to the medium at therequired rate. The acid solutions used to control pH may be sulfuricacid or hydrochloric acid. The base solutions used to control pH may besodium hydroxide, potassium hydroxide, or ammonium hydroxide.

Further, dissolved oxygen may be monitored in a culture medium by adissolved oxygen probe immersed in the culture medium. If dissolvedoxygen regulation is in effect, the oxygen level may be adjusted byincreasing or decreasing the stirring speed. The dissolved oxygen levelmay also be adjusted by increasing or decreasing the gas flow rate. Thegas may be compressed air, oxygen, or nitrogen.

Stir speed may also be monitored in a fermentation process. In examples,the stirrer motor may drive an agitator. The stirrer speed may be set ata consistent rpm throughout the fermentation or may be regulateddynamically to maintain a set dissolved oxygen level.

Additionally, turbidity may be monitored in a fermentation process. Inexamples, cell density may be measured using a turbidity probe.Alternatively, cell density may be measured by taking samples from thebioreactor and analyzing them in a spectrophotometer. Further, samplesmay be removed from the bioreactor at time intervals through a sterilesampling apparatus. The samples may be analyzed for alkaloids producedby the host cells. The samples may also be analyzed for othermetabolites and sugars, the depletion of culture medium components, orthe density of cells.

In another example, a feed stock parameter may be monitored during afermentation process. In particular, feed stocks including sugars andother carbon sources, nutrients, and cofactors that may be added intothe fermentation using an external pump. Other components may also beadded during the fermentation including, without limitation, anti-foam,salts, chelating agents, surfactants, and organic liquids.

Any convenient codon optimization techniques for optimizing theexpression of heterologous polynucleotides in host cells may be adaptedfor use in the subject host cells and methods, see e.g., Gustafsson, C.et al. (2004) Trends Biotechnol, 22, 346-353, which is incorporated byreference in its entirety.

The subject method may also include adding a starting compound to thecell culture. Any convenient methods of addition may be adapted for usein the subject methods. The cell culture may be supplemented with asufficient amount of the starting materials of interest (e.g., asdescribed herein), e.g., a mM to μM amount such as between about 1-5 mMof a starting compound. It is understood that the amount of startingmaterial added, the timing and rate of addition, the form of materialadded, etc., may vary according to a variety of factors. The startingmaterial may be added neat or pre-dissolved in a suitable solvent (e.g.,cell culture media, water, or an organic solvent). The starting materialmay be added in concentrated form (e.g., 10× over desired concentration)to minimize dilution of the cell culture medium upon addition. Thestarting material may be added in one or more batches, or by continuousaddition over an extended period of time (e.g., hours or days).

Methods for Isolating Products from the Fermentation Medium

The subject methods may also include recovering the BIA of interest fromthe cell culture. Any convenient methods of separation and isolation(e.g., chromatography methods or precipitation methods) may be adaptedfor use in the subject methods to recover the BIA of interest from thecell culture. Filtration methods may be used to separate soluble frominsoluble fractions of the cell culture. In some cases, liquidchromatography methods (e.g., reverse phase HPLC, size exclusion, normalphase chromatography) may be used to separate the BIA of interest fromother soluble components of the cell culture. In some cases, extractionmethods (e.g., liquid extraction, pH based purification, etc.) may beused to separate the BIA of interest from other components of the cellculture.

The produced alkaloids may be isolated from the fermentation mediumusing methods known in the art. A number of recovery steps may beperformed immediately after (or in some instances, during) thefermentation for initial recovery of the desired product. Through thesesteps, the alkaloids (e.g., BIAs) may be separated from the cells,cellular debris and waste, and other nutrients, sugars, and organicmolecules may remain in the spent culture medium. This process may beused to yield a BIA-enriched product.

In an example, a product stream having a benzylisoquinoline alkaloid(BIA) product is formed by providing engineered yeast cells and afeedstock including nutrients and water to a batch reactor. Theengineered yeast cells may have at least one modification selected fromthe group consisting of: a feedback inhibition alleviating mutation in abiosynthetic enzyme gene native to the cell; a transcriptionalmodulation modification of a biosynthetic enzyme gene native to thecell; and an inactivating mutation in an enzyme native to the cell. Whenthe engineered yeast cells are within the batch reactor, the engineeredyeast cells may be subjected to fermentation. In particular, theengineered yeast cells may be subjected to fermentation by incubatingthe engineered yeast cells for a time period of at least about 5 minutesto produce a solution comprising the BIA product and cellular material.Once the engineered yeast cells have been subjected to fermentation, atleast one separation unit may be used to separate the BIA product fromthe cellular material to provide the product stream comprising the BIAproduct. In particular, the product stream may include the BIA productas well as additional components, such as a clarified yeast culturemedium. Additionally, a BIA product may comprise one or more BIAs ofinterest, such as one or more BIA compounds.

Different methods may be used to remove cells from a bioreactor mediumthat include a BIA of interest. In examples, cells may be removed bysedimentation over time. This process of sedimentation may beaccelerated by chilling or by the addition of fining agents such assilica. The spent culture medium may then be siphoned from the top ofthe reactor or the cells may be decanted from the base of the reactor.Alternatively, cells may be removed by filtration through a filter, amembrane, or other porous material. Cells may also be removed bycentrifugation, for example, by continuous flow centrifugation or byusing a continuous extractor.

If some valuable BIAs of interest are present inside the cells, thecells may be permeabilized or lysed and the cell debris may be removedby any of the methods described above. Agents used to permeabilize thecells may include, without limitation, organic solvents (e.g., DMSO) orsalts (e.g., lithium acetate). Methods to lyse the cells may include theaddition of surfactants such as sodium dodecyl sulfate, or mechanicaldisruption by bead milling or sonication.

BIAs of interest may be extracted from the clarified spent culturemedium through liquid-liquid extraction by the addition of an organicliquid that is immiscible with the aqueous culture medium. Examples ofsuitable organic liquids include, but are not limited to, isopropylmyristate, ethyl acetate, chloroform, butyl acetate, methylisobutylketone, methyl oleate, toluene, oleyl alcohol, ethyl butyrate. Theorganic liquid may be added to as little as 10% or as much as 100% ofthe volume of aqueous medium.

In some cases, the organic liquid may be added at the start of thefermentation or at any time during the fermentation. This process ofextractive fermentation may increase the yield of BIAs of interest fromthe host cells by continuously removing BIA precursors or BIAs to theorganic phase.

Agitation may cause the organic phase to form an emulsion with theaqueous culture medium. Methods to encourage the separation of the twophases into distinct layers may include, without limitation, theaddition of a demulsifier or a nucleating agent, or an adjustment of thepH. The emulsion may also be centrifuged to separate the two phases, forexample, by continuous conical plate centrifugation.

Alternatively, the organic phase may be isolated from the aqueousculture medium so that it may be physically removed after extraction.For example, the solvent may be encapsulated in a membrane.

In examples, BIAs of interest may be extracted from a fermentationmedium using adsorption methods. In particular, BIAs of interest may beextracted from clarified spent culture medium by the addition of a resinsuch as Amberlite® XAD4 or another agent that removes BIAs byadsorption. The BIAs of interest may then be released from the resinusing an organic solvent. Examples of suitable organic solvents include,but are not limited to, methanol, ethanol, ethyl acetate, or acetone.

BIAs of interest may also be extracted from a fermentation medium usingfiltration. At high pH, the BIAs of interest may form a crystalline-likeprecipitate in the bioreactor. This precipitate may be removed directlyby filtration through a filter, membrane, or other porous material. Theprecipitate may also be collected by centrifugation and/or decantation.

The extraction methods described above may be carried out either in situ(in the bioreactor) or ex situ (e.g., in an external loop through whichmedia flows out of the bioreactor and contacts the extraction agent,then is recirculated back into the vessel). Alternatively, theextraction methods may be performed after the fermentation is terminatedusing the clarified medium removed from the bioreactor vessel.

Methods for Purifying Products from Alkaloid-Enriched Solutions

Subsequent purification steps may involve treating the post-fermentationBIA precursor- or BIA-enriched product using methods known in the art torecover individual product species of interest to high purity.

In one example, BIA precursors or BIAs extracted in an organic phase maybe transferred to an aqueous solution. In some cases, the organicsolvent may be evaporated by heat and/or vacuum, and the resultingpowder may be dissolved in an aqueous solution of suitable pH. In afurther example, the BIA precursors or BIAs may be extracted from theorganic phase by addition of an aqueous solution at a suitable pH thatpromotes extraction of the BIA precursors or BIAs into the aqueousphase. The aqueous phase may then be removed by decantation,centrifugation, or another method.

The BIA precursor- or BIA-containing solution may be further treated toremove metals, for example, by treating with a suitable chelating agent.The BIA precursor- or BIA-containing solution may be further treated toremove other impurities, such as proteins and DNA, by precipitation. Inone example, the BIA precursor- or BIA-containing solution is treatedwith an appropriate precipitation agent such as ethanol, methanol,acetone, or isopropanol. In an alternative example, DNA and protein maybe removed by dialysis or by other methods of size exclusion thatseparate the smaller alkaloids from contaminating biologicalmacromolecules.

In further examples, the BIA precursor-, BIA-, or modifiedBIA-containing solution may be extracted to high purity by continuouscross-flow filtration using methods known in the art.

If the solution contains a mixture of BIA precursors or BIAs, it may besubjected to acid-base treatment to yield individual BIA of interestspecies using methods known in the art. In this process, the pH of theaqueous solution is adjusted to precipitate individual BIA precursors orBIAs at their respective pKas.

For high purity, small-scale preparations, the BIA precursors or BIAsmay be purified in a single step by liquid chromatography.

Yeast-Derived Alkaloid APIs Versus Plant-Derived APIs

The clarified yeast culture medium (CYCM) may contain a plurality ofimpurities. The clarified yeast culture medium may be dehydrated byvacuum and/or heat to yield an alkaloid-rich powder. This product isanalogous to the concentrate of poppy straw (CPS) or opium, which isexported from poppy-growing countries and purchased by APImanufacturers. For the purposes of this invention, CPS is arepresentative example of any type of purified plant extract from whichthe desired alkaloids product(s) may ultimately be further purified.Tables 2 and 3 highlight the impurities in these two products that maybe specific to either CYCM or CPS or may be present in both. Byanalyzing a product of unknown origin for a subset of these impurities,a person of skill in the art could determine whether the productoriginated from a yeast or plant production host.

API-grade pharmaceutical ingredients are highly purified molecules. Assuch, impurities that could indicate the plant- or yeast-origin of anAPI (such as those listed in Tables 2 and 3) may not be present at thatAPI stage of the product. Indeed, many of the API products derived fromyeast strains of the present invention may be largely indistinguishablefrom the traditional plant-derived APIs. In some cases, however,conventional alkaloid compounds may be subjected to chemicalmodification using chemical synthesis approaches which may show up aschemical impurities in plant-based products that require such chemicalmodifications. For example, chemical derivatization may often result ina set of impurities related to the chemical synthesis processes. Incertain situations, these modifications may be performed biologically inthe yeast production platform, thereby avoiding some of the impuritiesassociated with chemical derivation from being present in theyeast-derived product. In particular, these impurities from the chemicalderivation product may be present in an API product that is producedusing chemical synthesis processes but may be absent from an API productthat is produced using a yeast-derived product. Alternatively, if ayeast-derived product is mixed with a chemically derived product, theresulting impurities may be present but in a lesser amount than would beexpected in an API that only or primarily contains chemically derivedproducts. In this example, by analyzing the API product for a subset ofthese impurities, a person of skill in the art could determine whetherthe product originated from a yeast production host or the traditionalchemical derivatization route.

Non-limiting examples of impurities that may be present inchemically-derivatized morphinan APIs but not in biosynthesized APIsinclude a codeine-O(6)-methyl ether impurity in API codeine;8,14-dihydroxy-7,8-dihydrocodeinone in API oxycodone; andtetrahydrothebaine in API hydrocodone. The codeine-O(6)-methyl ether maybe formed by chemical over-methylation of morphine. The8,14-dihydroxy-7,8-dihydrocodeinone in API oxycodone may be formed bychemical over-oxidation of thebaine. Additionally, thetetrahydrothebaine in API hydrocodone may be formed by chemicalover-reduction of thebaine.

However, in the case where the yeast-derived compound and theplant-derived compound are both subjected to chemical modificationthrough chemical synthesis approaches, the same impurities associatedwith the chemical synthesis process may be expected in the products. Insuch a situation, the starting material (e.g., CYCM or CPS) may beanalyzed as described above.

Methods of Engineering Host Cells

Also included are methods of engineering host cells for the purpose ofproducing BIAs of interest or precursors thereof. Inserting DNA intohost cells may be achieved using any convenient methods. The methods areused to insert the heterologous coding sequences into the host cellssuch that the host cells functionally express the enzymes and convertstarting compounds of interest into product BIAs of interest.

Any convenient promoters may be utilized in the subject host cells andmethods. The promoters driving expression of the heterologous codingsequences may be constitutive promoters or inducible promoters, providedthat the promoters are active in the host cells. The heterologous codingsequences may be expressed from their native promoters, or non-nativepromoters may be used. Such promoters may be low to high strength in thehost in which they are used. Promoters may be regulated or constitutive.In certain embodiments, promoters that are not glucose repressed, orrepressed only mildly by the presence of glucose in the culture medium,are used. Promoters of interest include but are not limited to,promoters of glycolytic genes such as the promoter of the B. subtilistsr gene (encoding the promoter region of the fructose bisphosphatealdolase gene) or the promoter from yeast S. cerevisiae gene coding forglyceraldehyde 3-phosphate dehydrogenase (GPD, GAPDH, or TDH3), the ADH1promoter of baker's yeast, the phosphate-starvation induced promoterssuch as the PHOS promoter of yeast, the alkaline phosphatase promoterfrom B. licheniformis, yeast inducible promoters such as Gal1-10, Gal1,GalL, GalS, repressible promoter Met25, tetO, and constitutive promoterssuch as glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcoholdehydrogenase promoter (ADH), translation-elongation factor-1-α promoter(TEF), cytochrome c-oxidase promoter (CYC1), MRP7 promoter, etc.Autonomously replicating yeast expression vectors containing promotersinducible by hormones such as glucocorticoids, steroids, and thyroidhormones may also be used and include, but are not limited to, theglucorticoid responsive element (GRE) and thyroid hormone responsiveelement (TRE). These and other examples are described U.S. Pat. No.7,045,290, which is incorporated by reference, including the referencescited therein. Additional vectors containing constitutive or induciblepromoters such as a factor, alcohol oxidase, and PGH may be used.Additionally any promoter/enhancer combination (as per the EukaryoticPromoter Data Base EPDB) could also be used to drive expression ofgenes. Any convenient appropriate promoters may be selected for the hostcell, e.g., E. coli. One may also use promoter selection to optimizetranscript, and hence, enzyme levels to maximize production whileminimizing energy resources.

Any convenient vectors may be utilized in the subject host cells andmethods. Vectors of interest include vectors for use in yeast and othercells. The types of yeast vectors may be broken up into 4 generalcategories: integrative vectors (YIp), autonomously replicating highcopy-number vectors (YEp or 2μ plasmids), autonomously replicating lowcopy-number vectors (YCp or centromeric plasmids) and vectors forcloning large fragments (YACs). Vector DNA is introduced intoprokaryotic or eukaryotic cells via any convenient transformation ortransfection techniques.

Utility

The host cells and methods of the invention, e.g., as described above,find use in a variety of applications. Applications of interest include,but are not limited to: research applications and therapeuticapplications. Methods of the invention find use in a variety ofdifferent applications including any convenient application where theproduction of BIAs is of interest.

The subject host cells and methods find use in a variety of therapeuticapplications. Therapeutic applications of interest include thoseapplications in which the preparation of pharmaceutical products thatinclude BIAs is of interest. The host cells described herein producebenzylisoquinoline alkaloid precursors (BIA precursors) and BIAs ofinterest. Reticuline is a major branch point intermediate of interest inthe synthesis of BIAs including engineering efforts to produce endproducts such as opioid products. The subject host cells may be utilizedto produce BIA precursors from simple and inexpensive starting materialsthat may find use in the production of BIAs of interest, includingreticuline, and BIA end products. As such, the subject host cells finduse in the supply of therapeutically active BIAs or precursors thereof.

In some instances, the host cells and methods find use in the productionof commercial scale amounts of BIAs or precursors thereof where chemicalsynthesis of these compounds is low yielding and not a viable means forlarge-scale production. In certain cases, the host cells and methods areutilized in a fermentation facility that would include bioreactors(fermenters) of e.g., 5,000-200,000 liter capacity allowing for rapidproduction of BIAs of interest or precursors thereof for therapeuticproducts. Such applications may include the industrial-scale productionof BIAs of interest from fermentable carbon sources such as cellulose,starch, and free sugars.

The subject host cells and methods find use in a variety of researchapplications. The subject host cells and methods may be used to analyzethe effects of a variety of enzymes on the biosynthetic pathways of avariety of BIAs of interest or precursors thereof. In addition, the hostcells may be engineered to produce BIAs or precursors thereof that finduse in testing for bioactivity of interest in as yet unproventherapeutic functions. In some cases, the engineering of host cells toinclude a variety of heterologous coding sequences that encode for avariety of enzymes elucidates the high yielding biosynthetic pathwaystowards BIAs of interest or precursors thereof. In certain cases,research applications include the production of precursors fortherapeutic molecules of interest that may then be further chemicallymodified or derivatized to desired products or for screening forincreased therapeutic activities of interest. In some instances, hostcell strains are used to screen for enzyme activities that are ofinterest in such pathways, which may lead to enzyme discovery viaconversion of BIA metabolites produced in these strains.

The subject host cells and methods may be used as a production platformfor plant specialized metabolites. The subject host cells and methodsmay be used as a platform for drug library development as well as plantenzyme discovery. For example, the subject host cells and methods mayfind use in the development of natural product based drug libraries bytaking yeast strains producing interesting scaffold molecules, such asprotopine, and further functionalizing the compound structure throughcombinatorial biosynthesis or by chemical means. By producing druglibraries in this way, any potential drug hits are already associatedwith a production host that is amenable to large-scale culture andproduction. As another example, these subject host cells and methods mayfind use in plant enzyme discovery. The subject host cells provide aclean background of defined metabolites to express plant EST librariesto identify new enzyme activities. The subject host cells and methodsprovide expression methods and culture conditions for the functionalexpression and increased activity of plant enzymes in yeast.

Kits and Systems

Aspects of the invention further include kits and systems, where thekits and systems may include one or more components employed in methodsof the invention, e.g., host cells, starting compounds, heterologouscoding sequences, vectors, culture medium, etc., as described herein. Insome embodiments, the subject kit includes a host cell (e.g., asdescribed herein), and one or more components selected from thefollowing: starting compounds, a heterologous coding sequence and/or avector including the same, vectors, growth feedstock, componentssuitable for use in expression systems (e.g., cells, cloning vectors,multiple cloning sites (MCS), bi-directional promoters, an internalribosome entry site (IRES), etc.), and a culture medium.

Any of the components described herein may be provided in the kits,e.g., host cells including one or more modifications, startingcompounds, culture medium, etc. A variety of components suitable for usein making and using heterologous coding sequences, cloning vectors andexpression systems may find use in the subject kits. Kits may alsoinclude tubes, buffers, etc., and instructions for use. The variousreagent components of the kits may be present in separate containers, orsome or all of them may be pre-combined into a reagent mixture in asingle container, as desired.

Also provided are systems for producing a BIA of interest, where thesystems may include engineered host cells including one or moremodifications (e.g., as described herein), starting compounds, culturemedium, a fermenter and fermentation equipment, e.g., an apparatussuitable for maintaining growth conditions for the host cells, samplingand monitoring equipment and components, and the like. A variety ofcomponents suitable for use in large scale fermentation of yeast cellsmay find use in the subject systems.

In some cases, the system includes components for the large scalefermentation of engineered host cells, and the monitoring andpurification of BIA compounds produced by the fermented host cells. Incertain embodiments, one or more starting compounds (e.g., as describedherein) are added to the system, under conditions by which theengineered host cells in the fermenter produce one or more desired BIAproducts or precursors thereof. In some instances, the host cellsproduce a BIA of interest (e.g., as described herein). In certain cases,the BIA products of interest are opioid products, such as codeine,neopine, morphine, neomorphine, hydrocodone, oxycodone, hydromorphone,dihydrocodeine, 14-hydroxycodeine, or dihydromorphine.

In some cases, the system includes means for monitoring and or analyzingone or more BIA compounds or precursors thereof produced by the subjecthost cells. For example, a LC-MS analysis system as described herein, achromatography system, or any convenient system where the sample may beanalyzed and compared to a standard, e.g., as described herein. Thefermentation medium may be monitored at any convenient times before andduring fermentation by sampling and analysis. When the conversion ofstarting compounds to BIA products or precursors of interest iscomplete, the fermentation may be halted and purification of the BIAproducts may be done. As such, in some cases, the subject systemincludes a purification component suitable for purifying the BIAproducts or precursors of interest from the host cell medium into whichit is produced. The purification component may include any convenientmeans that may be used to purify the BIA products or precursors offermentation, including but not limited to, silica chromatography,reverse-phase chromatography, ion exchange chromatography, HICchromatography, size exclusion chromatography, liquid extraction, and pHextraction methods. In some cases, the subject system provides for theproduction and isolation of BIA fermentation products of interestfollowing the input of one or more starting compounds to the system.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.), but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

EXPERIMENTAL Example I

A series of specific genetic modifications provide a biosyntheticprocess in Saccharomyces cerevisiae for the production of BIAs fromsimple, inexpensive feedstocks or precursor molecules. Methods forconstructing novel strains capable of producing the early BIA moleculesnorcoclaurine (NC) and norlaudanosoline (NL) from non-BIA precursors orsimple feedstocks are described. NC has never been reported as a productof microbial synthesis and is the natural precursor to all known BIAmolecules. Methods for manipulating the regulation of yeast biosyntheticpathways and for optimizing the production of aromatic amino acids andrelated BIA precursors are also described.

A. Tyrosine and Related BIA Precursor Overproducing Yeast Strains

Strains of S. cerevisiae are developed with improved flux through thearomatic amino acid biosynthesis pathway for the purposes of increasingintracellular concentrations of BIA precursor molecules includingtyrosine, 4-hydroxyphenylacetaldehyde (4-HPA),L-3,4-dihydroxyphenylalanine (L-DOPA), 3,4-dihydroxyphenylacetaldehyde(3,4-DHPA), and dopamine. These strains combine genetic modifications tothe yeast strain for the purpose of increasing carbon flux from centralmetabolism towards aromatic amino acid synthesis in general, towardstyrosine in particular, and include the introduction of key heterologousenzymes for the production of BIA precursor molecules not naturallyproduced by yeast. Genetic modifications are employed including theintroduction of feedback inhibition alleviating mutations to genesencoding native biosynthetic enzymes, tuning of transcriptionalregulation of native biosynthetic enzymes, deletion of genes encodingenzymes that divert precursor molecules away from the intended pathway,and introduction of heterologous enzymes for the conversion of naturallyendogenous molecules into non-native BIA precursor molecules.

Specific Description:

1.1) The biosynthetic pathway in the engineered strain incorporatesfeedback inhibition alleviating mutations (1.1.1) to the native yeastgene ARO4, which encodes a 3-deoxy-d-arabinose-heptulosonate-7-phosphate(DAHP) synthase, alone or in combination. This mutation (ARO4^(FBR)) isincorporated as a directed mutation to the native gene at the originallocus, as an additional copy introduced as a genetic integration at aseparate locus, or as an additional copy on an episomal vector such as a2μ or centromeric plasmid. FBR refers to feedback resistant mutants andmutations. The feedback inhibited copy of the DAHP synthase enzyme isunder the native yeast transcriptional regulation or is introduced withengineered constitutive or dynamic regulation of protein expression byplacing it under the control of a synthetic promoter.

1.1.1) ARO4^(FBR) mutations may include, for example, a substitution ofthe lysine residue at position 229 with a leucine (see e.g., Hartmann M,et al. (2003) Evolution of feedback-inhibited beta/alpha barrelisoenzymes by gene duplication and a single mutation. Proc Natl Acad SciUSA 100(3):862-867), a substitution of the glutamine residue at position166 with a lysine residue (see e.g., Fukuda K et al. (1992)Feedback-Insensitive Mutation of3-Deoxy-D-Arabino-Hepturosonate-7-Phosphate Synthase Caused by a SingleNucleotide Substitution of Aro4 Structural Gene inSaccharomyces-Cerevisiae. J Ferment Bioeng 74(2):117-119), or anadditional mutation conferring feedback inhibition selected from amutagenized library of microbial DHAP synthase mutants. Examples of suchselections include rescue of growth on o-fluoro-D,L-phenylalanine (seee.g., Fukuda et al. (1990) Breeding of Brewing Yeast Producing a LargeAmount of Beta-Phenylethyl Alcohol and Beta-Phenylethyl Acetate. AgrBiol Chem Tokyo 54(1):269-271) or growth of aro3 mutant yeast strains onmedia with excess tyrosine.

1.2) The biosynthetic pathway in the engineered strain incorporates afeedback inhibition alleviating mutation (1.2.1) to the native yeastgene ARO7, which encodes the enzyme chorismate mutase. This mutation(ARO7^(FBR)) is incorporated as a directed mutation to the native geneat the original locus, as an additional copy introduced as a geneticintegration at a separate locus, or as an additional copy on an episomalvector such as a 2μ or centromeric plasmid. The feedback inhibited copyof the chorismate mutase enzyme is under the native yeasttranscriptional regulation or is introduced with engineered constitutiveor dynamic regulation of protein expression by placing it under thecontrol of a synthetic promoter.

1.2.1) ARO7^(FBR) mutant alleles may include, for example, asubstitution of the threonine residue at position 226 with an isoleucine(see e.g., Schmidheini et al. (1989) A Single Point Mutation Results ina Constitutively Activated and Feedback-Resistant Chorismate Mutase ofSaccharomyces-Cerevisiae. J Bacteriol 171(3):1245-1253) or an additionalmutation conferring feedback inhibition selected from a mutagenizedlibrary of microbial chorismate mutase mutants. Examples of suchselections include assays for 5-methyltryptophan sensitivity orincreased production of melanin pigments in strains expressingheterologous tyrosinase enzymes (1.9) in the absence of externally fedtyrosine.

1.3) The biosynthetic pathway in the engineered strain incorporates theintroduction of a strong promoter element (such as GPD1, TEF1, etc) forthe overexpression of the native yeast gene ARO10, which encodes anenzyme with hydroxyphenylpyruvate decarboxylase activity. This geneticmodification is incorporated as a directed swapping of the nativepromoter DNA sequence at the original locus, as an additional copy ofthe gene under new transcriptional regulation introduced as a geneticintegration at a separate locus, or as an additional copy on an episomalvector such as a 2μ or centromeric plasmid.

1.4) The biosynthetic pathway in the engineered strain incorporates theintroduction of a strong promoter element (such as GPD1, TEF1, etc) forthe overexpression of the native yeast gene ARO9, which encodes anenzyme with hydroxyphenylpyruvate/glutamic acid transaminase activity.This genetic modification is incorporated as a directed swapping of thenative promoter DNA sequence at the original locus, as an additionalcopy of the gene under new transcriptional regulation introduced as agenetic integration at a separate locus, or as an additional copy on anepisomal vector such as a 2μ or centromeric plasmid.

1.5) The biosynthetic pathway in the engineered strain incorporates theintroduction of a strong promoter element (such as GPD1, TEF1, etc) forthe overexpression of the native yeast gene TKL, which encodes an enzymewith transketolase activity. This genetic modification is incorporatedas a directed swapping of the native promoter DNA sequence at theoriginal locus, as an additional copy of the gene under newtranscriptional regulation introduced as a genetic integration at aseparate locus, or as an additional copy on an episomal vector such as a2μ or centromeric plasmid.

1.6) The biosynthetic pathway in the engineered strain is improved bythe incorporation of a deletion or inactivating mutation of the nativeyeast gene ZWF1, which encodes an enzyme with glucose-6-phosphatedehydrogenase activity.

1.7) The biosynthetic pathway in the engineered strain is improved bythe incorporation of one or more deletion(s) or inactivating mutation(s)of known native alcohol dehydrogenase enzymes, such as the enzymesencoded by the genes ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, or SFA1.

1.8) The biosynthetic pathway in the engineered strain is improved bythe incorporation of one or more deletion(s) or inactivating mutation(s)of known native aldehyde oxidoreductases, such as ALD2, ALD3, ALD4,ALD5, or ALD6.

1.9) The biosynthetic pathway incorporates a heterologous enzyme for theconversion of tyrosine to L-DOPA. This enzyme may be from one of severalclasses, including, but not limited to bacterial tyrosinases, eukaryotictyrosinases, and tyrosine hydroxylases (Table 1). The gene for thisenzyme is incorporated as a genetic integration or on an episomal vectorsuch as a 2μ or centromeric plasmid. This L-DOPA producing enzyme isintroduced with engineered constitutive or dynamic regulation of proteinexpression by placing it under the control of a synthetic promoter.

1.9.1) In a biosynthetic pathway using a tyrosine hydroxylase enzyme forthe conversion of tyrosine to L-DOPA, additional expression of genesencoding enzymes for the synthesis and recycling of the pterin cofactortetrahydrobiopterin (BH4) and its derivatives are incorporated into theengineered strain in support of the activity of the tyrosine hydroxylaseenzyme. These enzymes include GTP cyclohydrolase,6-pyruvoyltetrahydropterin synthase, sepiapterin reductase,4a-hydroxytetrahydrobiopterin dehydratase, and quinoid dihydropteridinereductase (Table 1). The genes for these enzymes are incorporated as agenetic integration or on an episomal vector such as a 2μ or centromericplasmid. These BH4 synthesis and recycling enzymes are introduced withengineered constitutive or dynamic regulation of protein expression byplacing it under the control of a synthetic promoter.

1.10) The biosynthetic pathway incorporates a heterologous enzyme forthe decarboxylation of L-DOPA to produce dopamine. Enzymes with thisactivity are encoded by a genes from a variety of organisms includingbacteria, plants, and mammals. Examples include Pseudomonas putida DOPAdecarboxylase (PpDODC), Rattus norvegicus DOPA decarboxylase (RnDODC),and Papaver somniferum tyrosine/DOPA decarboxylase (PsTYDC) (Table 1).The gene for this enzyme is incorporated as a genetic integration or onan episomal vector such as a 2μ or centromeric plasmid. This dopamineproducing enzyme is introduced with engineered constitutive or dynamicregulation of protein expression by placing it under the control of asynthetic promoter.

1.11) A biosynthetic pathway for the production of 3,4-DHPA incorporatesa heterologous enzyme for the oxidation of dopamine to 3,4-DHPA.Examples of genes encoding this enzyme that may be used in the straininclude human monoamine oxidase A (hMAOA), E. coli monoamine oxidase(EcMAO), and Micrococcus luteus monoamine oxidase (MIMAO) (Table 1). Thegene for this enzyme is incorporated as a genetic integration or on anepisomal vector such as a 2μ or centromeric plasmid. This 3,4-DHPAproducing enzyme is introduced with engineered constitutive or dynamicregulation of protein expression by placing it under the control of asynthetic promoter.

1.11.1) Strains for the production of NC require dopamine and 4-HPA,while strains for the production of NL require dopamine and 3,4-DHPA,but not 4-HPA. A specific modification for the conversion of an NCproducing strain into an NL producing strain is the integration of a MAOgene into the yeast genome at the locus encoding the native yeast geneARO10. This combines a deletion of the native yeast enzyme responsiblefor converting a tyrosine biosynthetic precursor to 4-HPA with theintroduction of the enzyme capable of converting dopamine to 3,4-DHPA.

1.11.2) Yeast strains are constructed with a gene that expresses a MAOenzyme (1.11) under the control of an inducible promoter. When thestrain is grown in the presence of the inducer it may catalyze theconversion of dopamine to 3,4-DHPA, in the absence of inducer the strainonly produces 4-HPA.

1.11.2.1) Yeast strains are constructed with inducible MAO expression(1.11.2), where the inducible system also contains a DNA binding proteintargeted to the promoter regulating the ARO10 gene (1.3). The syntheticpromoter controlling ARO10 is therefore repressed when the promotercontrolling the MAO gene is activated and ARO10 is only expressed whenthe MAO gene is not transcriptionally active. This system allows for theconstruction of a single strain that conditionally only produces theprecursors for NC or NL.

B. NC-Producing Yeast Strains

Methods are developed to produce the BIA molecule NC in yeast anddemonstrate a first system for microbial synthesis of NC. With theengineered strains described herein, NC is produced and accumulated forits own value or combined with a biosynthetic pathway of additionalheterologous enzymes for the complete synthesis of downstream BIAs.

Specific Description:

2.1) Yeast strains are grown in liquid culture to a high cellconcentration before back diluting to intermediate concentrations (asmeasured by optical density or OD) in defined media containing highconcentrations of dopamine. The media components only need to satisfyconditions for growth of the strains; various growth feedstocks are used(for example, different sugars, nitrogen sources). The NC produced bythese yeast strains is excreted by the yeast cells and is measurable inthe spent media. Additional NC retained by cells is recovered via celllysis and extraction from the lysate.

2.2) Yeast strains containing various combinations of the modificationsas described in (1.1-1.8) substantially improve NC production from thatmeasurable in unmodified strains in fed dopamine assays as describedabove (2.1). In conditions where no extracellular tyrosine is availablein the yeast media, modifications described (1.1-1.8) provide forproduction of NC from fed dopamine; under these conditions the NCproduction from unmodified yeast strains is most often undetectable.

2.3) Yeast strains that produce NC when containing the modification asdescribed in (1.10) and grown as described in (2.1) when the additionalBIA precursor added to media is L-DOPA instead of dopamine.

2.3.1) Yeast strains as described in (2.3) containing variouscombinations of the modifications as described in (1.1-1.8)substantially improve production of NC.

2.4) Yeast strains that produce NC when containing both the heterologousenzymes for conversion of tyrosine to dopamine (1.9, 1.10) alongsidevarious combinations of modifications described above (1.1-1.8) andgrown in media without supplementation of tyrosine, L-DOPA, or dopamine.This specific example constitutes complete synthesis of NC by the strainfrom simple carbon and nitrogen sources.

2.5) Yeast strains are modified and cultured as described above(2.1-2.4) where the biosynthetic pathway includes the incorporation ofthe heterologous enzyme NCS, or truncated versions of the NCS enzyme,for the stereospecific catalysis of the reaction condensing dopamine and4-HPA for S-NC production. This enzyme may originate from one of severalplants, such as Papaver somniferum, Coptis japonica, and Thalicitumflavum (Table 1). The gene for this enzyme is incorporated as a geneticintegration or on an episomal vector such as a 2μ or centromericplasmid. This S-NC producing enzyme is introduced with engineeredconstitutive or dynamic regulation of protein expression by placing itunder the control of a synthetic promoter. The NC ultimately producedwill be an enantiomeric mixture with bias towards the S-stereoisomer.

2.6) Yeast strains are modified and cultured as described above(2.1-2.5) where the biosynthetic pathway includes the incorporation ofthe heterologous enzyme norcoclaurine 6-O-methyltransferase (6OMT) forthe catalysis of the reaction producing coclaurine. 6OMT may originatefrom one of several plants, such as Papaver somniferum, Coptis japonica,and Thalicitum flavum (Table 1).

2.7) Yeast strains are modified and cultured as described above(2.1-2.5) where the biosynthetic pathway includes the incorporation ofthe heterologous enzymes 6OMT and coclaurine N-methyltransferase (CNMT)for the catalysis of the reactions producing coclaurine andN-methylcoclaurine. CNMT may originate from one of several plants, suchas Papaver somniferum, Coptis japonica, and Thalicitum flavum (Table 1).

2.8) Yeast strains are modified and cultured as described above(2.1-2.5) where the biosynthetic pathway includes the incorporation ofthe heterologous enzymes 6OMT, CNMT, CYP80B1, and cytochrome P450reductase (CPR) for the catalysis of the reactions producing coclaurine,N-methylcoclaurine, and 3′hydroxy-N-methyl-coclaurine. CYP80B1 mayoriginate from one of several plants, such as Papaver somniferum orEschscholzia californica (Table 1).

2.9) Yeast strains are modified and cultured as described above(2.1-2.5) where the biosynthetic pathway includes the incorporation ofthe heterologous enzymes 6OMT, CNMT, CYP80B1, CPR, and3′hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT) for thecatalysis of the reactions producing coclaurine, N-methylcoclaurine,3′hydroxy-N-methyl-coclaurine, and reticuline. 4′OMT may originate fromone of several plants, such as Papaver somniferum, Coptis japonica, andThalicitum flavum (Table 1).

C. NL-Producing Yeast Strains

Methods are developed to produce the BIA molecule NL from yeast. Withthe engineered strains described herein, NL is produced and accumulatedfor its own value or combined with a biosynthetic pathway of furtherheterologous enzymes for the complete synthesis of downstream BIAs.

Specific Description:

3.1) Yeast strains containing modifications as described in (1.11,1.11.1-1.11.2, 1.11.2.1) are grown in liquid culture as described in(2.1) produce NL.

3.2) Yeast strains containing various combinations of gene deletions asdescribed in (1.7, 1.8) improve NL production from that measurable inunmodified strains in fed dopamine assays as described above (3.1).

3.3) Yeast strains that produce NL when containing the modifications asdescribed in (1.10, 1.11, 1.11.1-1.11.2, 1.11.2.1) and grown asdescribed in (3.1) when the additional BIA precursor is added to mediais L-DOPA instead of dopamine.

3.3.1) Yeast strains as described in (3.3) containing combinations ofgene deletions described in (1.7, 1.8) improve production of NL.

3.4) Yeast strains that produce NL when containing both the heterologousenzymes for conversion of tyrosine to dopamine (1.9, 1.10) and dopamineto 3,4-DHPA (1.11) alongside various combinations of modificationsdescribed above (1.1-1.8, 1.11.1) are grown in media withoutsupplementation of tyrosine, L-DOPA, or dopamine. This specific exampleconstitutes complete synthesis of NL by the strain from simple carbonand nitrogen sources.

3.5) Yeast strains modified and cultured as described above (3.1-3.4)where the biosynthetic pathway includes the incorporation of theheterologous enzyme NCS, or truncated versions of the NCS enzyme (Table1), for the stereospecific catalysis of the reaction condensing dopamineand 3,4-DHPA for S-NL production. This enzyme may originate from one ofseveral plants, such as Papaver somniferum, Coptis japonica, andThalicitum flavum (Table 1). The gene for this enzyme is incorporated asa genetic integration or on an episomal vector such as a 2μ orcentromeric plasmid. This S-NL producing enzyme is introduced withengineered constitutive or dynamic regulation of protein expression byplacing it under the control of a synthetic promoter. The NC ultimatelyproduced is an enantiomeric mixture with bias towards theS-stereoisomer.

3.6) Yeast strains are modified and cultured as described above(3.1-3.5) where the biosynthetic pathway includes the incorporation ofthe heterologous enzyme norcoclaurine 6-O-methyltransferase (6OMT) forthe catalysis of the reaction producing 3′hydroxycoclaurine. 6OMT mayoriginate from one of several plants, such as Papaver somniferum, Coptisjaponica, and Thalicitum flavum (Table 1).

3.7) Yeast strains are modified and cultured as described above(3.1-3.5) where the biosynthetic pathway includes the incorporation ofthe heterologous enzyme coclaurine N-methyltransferase (CNMT) for thecatalysis of the reaction producing laudanosoline. CNMT may originatefrom one of several plants, such as Papaver somniferum, Coptis japonica,and Thalicitum flavum (Table 1).

3.8) Yeast strains are modified and cultured as described above(3.1-3.5) where the biosynthetic pathway includes the incorporation ofthe heterologous enzymes 6OMT and CNMT for the catalysis of thereactions producing 3′hydroxycoclaurine, laudanosoline, and 3′hydroxyl-N-methyl-coclaurine.

3.9) Yeast strains are modified and cultured as described above(3.1-3.5) where the biosynthetic pathway includes the incorporation ofthe heterologous enzyme 3′hydroxy-N-methylcoclaurine4′-O-methyltransferase (4′OMT) for the catalysis of the reactionproducing 4′-O-methylnorlaudanosoline. 4′OMT may originate from one ofseveral plants, such as Papaver somniferum, Coptis japonica, andThalicitum flavum (Table 1).

3.10) Yeast strains are modified and cultured as described above(3.1-3.5) where the biosynthetic pathway includes the incorporation ofthe heterologous enzymes 6OMT and 4′OMT for the catalysis of thereactions producing 3′hydroxycoclaurine, 4′-O-methylnorlaudanosoline,and norreticuline.

3.11) Yeast strains are modified and cultured as described above(3.1-3.5) where the biosynthetic pathway includes the incorporation ofthe heterologous enzymes CNMT and 4′OMT for the catalysis of thereactions producing 4′-O-methylnorlaudanosoline, laudanosoline, and4′-O-methyllaudanosoline.

3.12) Yeast strains are modified and cultured as described above(3.1-3.5) where the biosynthetic pathway includes the incorporation ofthe heterologous enzymes 6OMT, CNMT, and 4′OMT for the catalysis of thereactions producing 3′hydroxycoclaurine, laudanosoline,4′-O-methylnorlaudanosoline, norreticuline,3′hydroxy-N-methylcoclaurine, 4′-O-methyllaudanosoline, and reticuline.

Example 2

Processing Methods from Fermentation to Purification to API

During the final stages of the fermentation, the organic liquid ethylacetate is added to the culture medium at 20% of the volume of theculture broth. At this time the pH of the culture medium may also beincreased to support the extraction of BIAs into the organic ethylacetate phase. The agitation from the stirrer and sparger causes theimmiscible ethyl acetate to form an emulsion with the aqueous medium. Atthe completion of the fermentation the stirrer and sparger are switchedoff and the aqueous and organic phases are allowed to separate. Ademulsifier or other chemical agent may be added to promote theseparation into two distinct phases. The cells settle to the bottom ofthe reactor and form a third layer. The three layers are separated bydecantation. The cell and aqueous culture medium phases are discarded.The BIA precursor- or BIA-containing organic phase is treated with heatand vacuum to remove the ethyl acetate. The resulting powder containingthe BIA precursors or BIAs is dissolved in acidified water. The solutionis purified by cross-flow filtration to yield a solution consisting onlyof morphinan alkaloids. The solution is then subjected to several roundsof acid-base extraction to remove individual morphinan alkaloidsaccording to their distinct solubility at known pH values. Eachprecipitate of morphinan alkaloid (e.g., codeine sulfate) is removed byfiltration and may be subject to further product polishing to yield anAPI-grade product.

Example 3

Processing Methods from Fermentation to Purification to API

The fermentation broth is centrifuged to remove cells and particulates.The remaining aqueous clarified culture medium is processed by a similarmethod used to extract BIAs from aqueous poppy straw extract in thekabay process (GB406,107). The clarified culture medium may be acidicdue to the fermentation processes. If not, the medium is acidified byaddition of sulfuric acid. The alkaloids are concentrated in theclarified culture medium by the application of a vacuum or heat. Thesolution is treated with ethanol to precipitate impurities includingproteins and DNA. Further treatment involves several rounds of acid-baseextraction and may include the addition of organic solvents to aid inextracting distinct BIA species into a second phase.

Example 4

Modification of Media to Improve BIA Production

BIA production titers from engineered yeast strains were improved bymodifying the culture media composition. For example, the media typescan vary in the media base (e.g., yeast peptone, yeast nitrogen base),carbon source (e.g., glucose, maltodextrin), and nitrogen source (e.g.,amino acids, ammonium sulfate, urea). Reticuline-producing yeast strains(as described in 2.9) were grown under varying media conditions andreticuline production assayed after 72 hours of growth at 30 C. Thehighest production of reticuline was observed in YNB, 2% maltodextrinwith 3 AGU/L, ammonium sulfate, and all amino acids (FIG. 17A).Maltodextrin is a polymer of glucose that can be broken down by amylase.When maltodextrin is used as the carbon source in yeast culture medium,the addition of amylase creates a slow release of glucose, therebymimicking fed-batch fermentation conditions. High concentration ofamylase results in faster release of glucose and is effectively a fasterglucose feed rate. In this example, the feed rate of glucose impactsproduct titers (FIG. 17B). The data demonstrate the highest titers ofreticuline in media comprising 4% maltodextrin with 3 AGU/L.

FIG. 1: Biosynthesis of Tyrosine and BIA Precursor Molecules

Schematic showing the biosynthetic pathway from glucose to tyrosine andother BIA precursors. Aromatic amino acid intermediates present innative yeast metabolism are written in black. Endogenous yeast enzymesare written in grey (apart from TYR, TyrH, DODC, and MAO). Heterologousenzymes include TYR, TyrH, DODC, and MAO. As described in (1.1, 1.2)wild-type yeast enzymes encoded by ARO4 and ARO7 are allostericallyinhibited by tyrosine, indicated here by the dotted grey line.Individual steps in the pentose phosphosphate pathway and glycolysis arenot explicitly detailed in this figure, although the genes TKL1 and ZWF1(targeted in 1.5, 1.6) are involved in the pentose phosphate pathway, asindicated.

FIG. 2: Effect of ZWF1 Knockout and TKL1 Over-Expression on PentosePhosphate Pathway (PPP)

Schematic detailing how modifications to TKL1 (1.5) and ZWF1 (1.6)affect the overall carbon flow through the pentose phosphate pathway inyeast when glucose is the primary carbon source. Panel A representswild-type carbon flow; Panel B represents the relative change in carbonflow in a modified strain.

FIG. 3A: Synthesis of NC from Precursor Molecules

NC is synthesized from one molecule of dopamine and one molecule of4-HPA via a Pictet-Spengler condensation reaction. This reaction mayoccur spontaneously to produce a racemic mixture of R- and S-NC. Thisreaction may alternatively be catalyzed by the plant enzyme NCS, whichproduces S-NC.

FIG. 3B: Synthesis of NL from Precursor Molecules

NL is synthesized from one molecule of dopamine and one molecule of3,4-DHPA via a Pictet-Spengler condensation reaction. This reaction mayoccur spontaneously to produce a racemic mixture of R- and S-NL. Whilethe natural product of NCS is NC, the enzyme has been shown to catalyzethe stereospecific production of S-NL (see e.g., Rueffer et al. (1981)(S)-Norlaudanosoline Synthase—the 1st Enzyme in the BenzylisoquinolineBiosynthetic-Pathway. Febs Lett 129(1):5-9).

Measurement of the BIA molecules is performed by LC-MS analysis, whereNC production (m/z=+272, 19.2 min retention time) and NL production(m/z=+288, 18.9 min retention time) were observed, with ion MS2fragmentation agreeing with both standards and published detectionmethods (see e.g., Schmidt et al. (2007) Poppy alkaloid profiling byelectrospray tandem mass spectrometry and electrospray FT-ICR massspectrometry after [ring-13C6]-tyramine feeding. Phytochemistry68(2):189-202).

FIG. 4: Effect of Four Genetic Modifications on NC Production withVarying Fed Tyrosine

NC production was demonstrated at several concentrations of fedtyrosine, including no fed tyrosine, in strains with targeted geneticmodifications. Wild-type strain, CEN.PK2, was integrated with constructsconferring one of four genetic changes (as described in 1.1-1.4):overexpression of ARO10 by promoter replacement with P_(TEF1),overexpression of ARO9 by promoter replacement with P_(TEF1),chromosomal integration of an ARO4^(FBR) allele, and chromosomalintegration of an ARO7^(FBR) allele. When incorporated alone, only theP_(TEF1)-ARO10 and ARO4^(FBR) increase production of NC. While boththese modifications increased NC production at all tyrosineconcentrations, the ARO^(FBR) integrated strain improved mostdrastically at zero fed tyrosine.

FIG. 5: NC Production with Combinations of Genetic Modifications

Some genetic modifications as described above (1.5, 1.6) improve NCproduction only in combination with the integration of the ARO4^(FBR)mutant (1.1). This figure shows four strains engineered with singlegenetic modifications, P_(TEF1)-ARO10 (1.3), P_(GPD)-TKL1 (1.5), ZWF1knockout (1.6), and ARO4^(FBR) (1.1), alongside three strainsconstructed with combinations of genetic modifications: Strain A(P_(GPD)-TKL1, ARO4^(FBR)), Strain B (ZWF1 knockout, ARO4^(FBR)), andStrain C (P_(GPD)-TKL1, ZWF1 knockout, ARO4^(FBR)). NC production isshown normalized to the WT strain, with Strain C exhibiting a five-foldincrease in NC production.

FIG. 6: NL Production in ALD/ADH Knockout Strains

NL production is improved by the deletion of competing yeast enzymes(1.7, 1.8) in a strain expressing human MAOA on a 2μ plasmid (1.11) andgrown in media containing dopamine. NL production is shown as titermeasured in spent media normalized to the WT (with hMAO, but with nodeletions). Improvements in production are as much as ten times WT NLproduction.

FIG. 7: Activity of a DODC Enzyme In Vivo

Yeast strains transformed with DNA to express Papaver somniferumtyrosine/DOPA decarboxylase may convert L-DOPA to dopamine in vivo.Strains harboring a 2μ plasmid were grown in selective media and thenback-diluted into media containing L-DOPA. Spent media was then measuredfor concentrations of L-DOPA (retention time 4.8 min, m/z=+198) anddopamine (retention time 4.2 min, m/z=+154).

FIG. 8

NC is produced as described in (2.1) in multiple wild-type yeast labstrains at varying tyrosine concentrations. Specifically, each yeaststrain is inoculated into separate liquid cultures and grown overnightto OD₆₀₀˜10, then back-diluted in YNB minimal media without tyrosine toan OD₆₀₀˜1 and grown for 3 hours. 100 μl of each culture was mixed into400 μl YNB media containing 100 mM dopamine and varying concentrationsof tyrosine; each strain was grown in each media condition in triplicatesamples. NC titer was measured from culture supernatant on an LC-MSinstrument detecting m/z+272 ion count in peaks as described by (seee.g., Schmidt et al. (2007) Poppy alkaloid profiling by electrospraytandem mass spectrometry and electrospray FT-ICR mass spectrometry after[ring-13C6]-tyramine feeding. Phytochemistry 68(2):189-202). The area ofeach peak was integrated to calculate a relative quantity of the NC ineach sample and the results were normalized to the ion count area inCEN.PK2 yeast culture with 0 mg/L tyrosine.

FIG. 9

NC is produced as described in (2.1) in multiple engineered yeaststrains at fed 100 mM dopamine and no tyrosine. These data weregenerated in a separate experiment from those in FIG. 5. The strainsCSY1031-1044 were engineered to contain combinations of the geneticmodifications described in (1.1-1.6); the labels underneath each strainname indicate which modifications were incorporated into each strain.Strain CSY1043 contains four genetic modifications to yeast nativemetabolism and exhibits the largest increase in NC titer above thewild-type yeast strain CEN.PK2.

FIG. 10

NC production as described in (2.1) for black diamonds and as describedin (2.3) for gray circles. Here NC was produced in an engineered yeaststrain (CSY1042) with the additional integration of the L-DOPAdecarboxylase PpDODC (1.10). In separate liquid cultures this yeaststrain was grown in YNB minimal media containing varying concentrationsof dopamine (black diamonds) and YNB minimal media containing L-DOPA(gray circles; cultures not fed dopamine). The solid black linerepresents a linear regression of the relationship between the measuredNC and fed dopamine. The peak area measurements for the L-DOPA fedsamples were plotted along the regression line for dopamine fed samplesto show an “equivalent fed dopamine” quantity for the cultures fedL-DOPA. L-DOPA media was mixed to achieve a target concentration of 10mM L-DOPA, however L-DOPA was not fully soluble at that concentration,and the effective concentration of dissolved L-DOPA is estimated to beapproximately 6 mM. Based on the average NC titers of the two L-DOPA fedyeast cultures the “equivalent fed dopamine” concentration isapproximately 50 mM or 8× the fed L-DOPA concentration (indicated by thegray dotted lines).

FIG. 11

Mammalian tyrosine hydroxylases (TyrHs) are capable of hydroxylatingtyrosine, but are dependent on the co-substrate tetrahydrobiopterin(BH4) for activity, as described in (1.9.1). During the catalysis oftyrosine to L-DOPA by TyrH, molecular oxygen is split and transferred totyrosine and BH4, as shown by reaction 1. BH4 is oxidized toBH4-4a-carbinolamine (4αOH-BH4). Two heterologous enzymes are expressedin yeast to synthesize BH4 from the folate synthesis pathwayintermediate, dihydroneopterin triphosphate. First,6-pyruvoyltetrahydropterin synthase (PTPS) converts dihydroneopterin toPTP (reaction 2), which is then reduced to BH4 by sepiapterin reductase(SepR, reaction 3). Two enzymes are responsible for the regeneration ofBH4 from its 4a-carbinolamine form. First, pterin-4a-carbinolaminedehydratase (PCD) catalyzes a loss of water reaction to formdihydrobioterin (reaction 4). Dihydrobiopterin is then reduced totetrahydrobiopterin by quinoid dihydropteridine reductase (QDHPR,reaction 5).

FIG. 12

Tyrosine hydroxylases expressed from yeast cells convert tyrosine toL-DOPA. Yeast strains transformed with plasmids carrying tyrosinehydroxylases from human (hTH2) and rat (RnTyrH) were grown in liquidmedia and then lysed in buffer containing tyrosine and the co-substrateBH4. After 6-hour incubations at 30° C., L-DOPA was measured in thelysate mixture by LC-MS. (A) LC-MS chromatogram confirms conversion oftyrosine to L-DOPA dependent on the presence of the co-substrate, BH4.(B) Fragmentation of the +198 m/z ion peak further confirms the presenceof L-DOPA in lysate samples. (See e.g., Lv et al. (2010) LC-MS-MSSimultaneous Determination of L-Dopa and its prodrug n-PentylHydrochloride in Rat Plasma. Chromatographia, 72(3/4), 239-243).

FIG. 13

Co-expression of tyrosine hydroxylase with a BH4 biosynthetic enzymeenables conversion of tyrosine to L-DOPA in yeast cell lysates.Engineered yeast strains integrated with constructs expressing rattyrosine hydroxylase (RnTyrH) and rat sepiapterin reductase (RnSepR)were grown in liquid media and then lysed in buffer containing tyrosine,NADPH, and the BH4 biosynthetic precursor, sepiapterin. Co-expression ofa TyrH with the BH4 biosynthesis gene provides for activity of thetyrosine hydroxylase in the absence of BH4, but in the presence of theBH4 precursor, sepiapterin.

FIG. 14

Biosynthetic pathways for the synthesis of BIA precursors andpre-reticuline BIAs from tyrosine through to reticuline. (A)Biosynthetic pathway from tyrosine to reticuline that goes through NC.(B) Biosynthetic pathway from tyrosine to reticuline that goes throughNL (depiction includes various methylated intermediates that can beproduced). * Note that although TyrH is depicted as catalyzing theconversion of L-tyrosine to L-DOPA, other enzymes including tyrosinasescan be used to perform this step as described in the specification.

FIG. 15

Engineered yeast strains produce NC-derived BIA molecules from L-DOPA inliquid culture. A copy of PpDODC was integrated into the engineeredyeast strain, CSY1039 (as described in FIG. 9), providing for theproduction of NC from L-DOPA (panel A, bottom chromatogram). Next a copyof the opium poppy 6-O-methyltransferase (Ps6OMT) gene was integratedinto this yeast strain to enable the production of coclaurine fromL-DOPA (panel A, middle chromatogram). Finally, a copy of both Ps6OMTand the opium poppy coclaurine-N-methyltransferase (PsCNMT) genes wereintegrated into the CSY1039 yeast strain carrying the PpDODC gene toenable the production of N-methylcoclaurine from L-DOPA (panel A, topchromatogram). Both the NC and coclaurine measurements matchedchromatograms from chemical standards. The production ofN-methylcoclaurine was further confirmed by matching the fragmentationpattern of the +300 m/z ion peak to patterns in published literature(Panel B). (see e.g., Schmidt et al. (2007) Poppy alkaloid profiling byelectrospray tandem mass spectrometry and electrospray FT-ICR massspectrometry after [ring-13C6]-tyramine feeding. Phytochemistry68(2):189-202).

FIG. 16

Engineered yeast strains produce norcoclaurine (as described in (2.5))and reticuline (as described in (2.9)) from sugar in liquid culture. (A)LC-MS/MS was used in MRM mode to detect norcoclaurine with thetransition 272 m/z to 107 m/z. The dashed line depicts a norcoclaurinestandard. Grey line shows norcoclaurine production in an engineeredyeast strain. The strain contains harbors the following modifications:ARO4^(FBR), ZWF1 knockout, GPD-TKL1 promoter replacement; and expressesthe following heterologous genes: wild type R. norvegicus tyrosinehydroxylase (RnTyrH; expressed from a low-copy plasmid), PpDODC(integrated into the chromosome), and BH4 biosynthesis and recyclinggenes (RnPTPS, RnSepR, RnPCD, RnQDHPR; integrated into the chromosome).The black line depicts norcoclaurine production in the same strain asdescribed for the grey line and also co-expressing T. flavumnorcoclaurine synthase (TfNCS; expressed from a low-copy plasmid). (B)LC-MS/MS was used in MRM mode to detect reticuline with the transition330 m/z to 137 m/z. The dashed line depicts a reticuline standard. Theblack line shows reticuline production in the strain described in (A,grey line) further engineered to express Ps6OMT, PsCNMT, EcCYP80B1,PsCPR, Ps4′OMT, and Coptis japonica norcoclaurine synthase (CjNCS;integrated into the chromosome).

FIG. 17

Effect of media composition on production of BIA products. Yeast strainsengineered to produce reticuline (as described in (2.9)) from sugar weregrown under various media conditions in liquid culture. LC-MS/MS wasused in MRM mode to detect reticuline with the transition 330 m/z to 137m/z in the growth media of strains grown for 72 hours at 30 C. (A)Reticuline production from yeast strain YA8 in a variety of mediaconditions. YPD, yeast peptone dextrose; YNB, yeast nitrogen base. (B)Optimization of reticuline titer while varying maltodextrinconcentration and amylase concentration with two reticuline-producingyeast strains, YA8 and YA22. AGU, amyloglucosidase unit.

FIG. 18

Effect of inactivating mutations in ADH and ALD enzymes on production ofBIA products. Combinations of inactivating mutations in ADH and ALDenzymes were incorporated into yeast strains engineered to producereticuline (as described in (2.9)). The enzymes with inactivatingmutations may include any combination of the following: ADH2, ADH3,ADH4, ADH5, ADH6, ADH7, ALD2, ALD3, ALD4, ALD5, or ALD6. In some cases,the cell will contain two, three, four, five, six, seven, eight, nine,ten, eleven, or more inactivating mutations. In certain cases, theenzymes containing inactivating mutations will be ADH2, ADH5, ADH6, andALD4. In certain cases the enzymes containing inactivating mutationswill be ADH2, ADH5, ADH6, ALD2, and ALD3. In some cases multiple ADH andALD inactivating mutations will increase BIA product titer. Combinationsof inactivating mutations in four enzymes (ADH2, ADH5, ADH6, ALD4) andfive enzymes (ADH2, ADH5, ADH6, ALD2, ALD3) increases the production ofreticuline from the reticuline-producing yeast strain (YA8). Yeaststrains engineered to produce reticuline (as described in (2.9)) fromsugar were grown in liquid culture for 72 hours at 30 C. LC-MS/MS wasused in MRM mode to detect reticuline with the transition 330 m/z to 137m/z in the growth media.

TABLE 1 Genes of interest as components of the engineered metabolicpathways Coding Specific Source Engineered sequence descrip- EnzymeAbbrev. Catalyzed reactions organisms regulation changes Genbank # tionref. 3-deoxy-d- ARO4, erythrose-4-phosphate + Saccharomyces native,Feedback CAA85212.1 1.1 arabinose- DHAP PEP → DHAP cerevisiaeconstitutive, inhibition heptulosonate- synthase (EC 2.5.1.54) syntheticresistant 7-phosphate regulation mutation, synthase K229L, Q166KChorismate mutase ARO7 chorismate → prephenate Saccharomyces native,Feedback NP_015385.1 1.2 (EC 5.4.99.5) cerevisiae constitutive,inhibition synthetic resistant regulation mutation, T226I PhenylpyruvateARO10 hydroxyphenylpyruvate → Saccharomyces constitutive NP_010668.3 1.3decarboxylase 4HPA (EC 4.1.1.80) cerevisiae overexpression, syntheticregulation Aromatic ARO9 hydroxyphenylpyruvate + Saccharomycesconstitutive AEC14313.1 1.4 aminotransferase glutamate → tyrosine +cerevisiae overexpression, alpha-ketogluterate synthetic (EC 2.6.1.57)regulation Transketolase TKL1 fructose-6-phosphate + Saccharomycesconstitutive NP_015399.1 1.5 glyceraldehyde-3- cerevisiaeoverexpression, phosphate ↔ xylulose-5- synthetic phosphate +erythrose-4- regulation phosphate (EC 2.2.1.1) Glucose-6- ZWF1glucose-6-phosphate → 6- Saccharomyces full deletion of CAA96146.1 1.6phosphate phosphogluconolactone cerevisiae coding region dehydrogenase(EC 1.1.1.49) Alcohol ADH2-7, 4HPA → tyrosol Saccharomyces full deletionof NP_014032.1, 1.7 dehydrogenase SFA1 (EC 1.1.1.90) cerevisiae codingregion AAT93007.1, NP_011258.2, NP_009703.3, NP_014051.3, NP_010030.1,NP_010113.1 Aldehyde oxidase ALD2-6 4HPA → Saccharomyces full deletionof NP_013893.1, 1.8 hydroxyphenylacetic acid cerevisiae coding regionNP_013892.1, (EC 1.2.1.39) NP_015019.1, NP_010996.2, NP_015264.1Tyrosinase TYR tyrosine → L-DOPA, L- Ralstonia constitutive NP_518458.1,1.9 DOPA → dopaquinone solanacearum, overexpression, AJ223816, (EC1.14.18.1) Agaricus synthetic bisporus regulation Tyrosine TyrH tyrosine→ L-DOPA Homo sapiens, constitutive NM 012740, 1.9 hydroxylase (EC1.14.16.2) Rattus overexpression, NM 000240, norvegicus, synthetic Musmusculus regulation GTP cyclohydrolase FOL2 GTP → dihydroneopterinSaccharomyces native CAA97297.1, 1.9.1 triphosphate (EC 3.5.4.16)cerevisiae, regulation, NP_001019195.1, Homo sapiens, constitutiveNP_032128.1 Mus musculus overexpression, synthetic regulation 6-pyruvoylPTPS dihydroneopterin Rattus constitutive AAH59140.1, 1.9.1 tetrahydro-triphosphate → PTP norvegicus, overexpression, BAA04224.1, biopterin(PTP) (EC 4.2.3.12) Homo sapiens, synthetic AAH29013.1 synthase Musmusculus regulation Sepiapterin SepR PTP → BH4 Rattus constitutiveNP_062054.1, 1.9.1 reductase (EC 1.1.1.153) norvegicus, overexpression,NP_003115.1, Homo sapiens, synthetic NP_035597.2 Mus musculus regulation4a- PCD 4a- Rattus constitutive NP_001007602.1, 1.9.1 hydroxytetra-hydroxytetrahydro- norvegicus, overexpression, AAB25581.1,hydrobiopterin biopterin → Homo sapiens, synthetic NP_079549.1(pterin-4α- H2O + quinoid Mus musculus regulation carbinolamine)dihydropteridine dehydratase (EC 4.2.1.96) Quinoid QDHPR quinoiddihydropteridine → Rattus constitutive AAH72536.1, 1.9.1dihydropteridine BH4 (EC 1.5.1.34) norvegicus, overexpression,NP_000311.2, reductase Homo sapiens, synthetic AAH02107.1 Mus musculusregulation L-DOPA DODC L-DOPA → dopamine Pseudomonas constitutiveAE015451.1, 1.10 decarboxylase (EC 4.1.1.28) putida, overexpression,NP_001257782.1 Rattus synthetic norvegicus regulation Tyrosine/DOPA TYDCL-DOPA → dopamine Papaver constitutive 1.10 decarboxylase (EC 4.1.1.28)somniferum overexpression, synthetic regulation Monoamine MAO dopamine →3,4-DHPA E. coli, Homo constitutive J03792, D2367, 1.11, oxidase (EC1.4.3.4) sapiens, overexpression, AB010716.1 1.11.1 Micrococcussynthetic luteus regulation Norcoclaurine NCS 4HPA + dopamine → S-Coptis japonica, constitutive N-terminal BAF45337.1, 2.5, 3.5 synthasenorcoclaurine (EC 4.2.1.78) Papaver truncation ACI45396.1, 3,4-DHPA +dopamine → somniferum, ACO90258.1, S- norlaudanosoline PapverACO90247.1, bracteatum, AEB71889.1 Thalicitum flavum, Corydalis saxicolaNorcoclaurine 6-O- 6OMT Norcoclaurine → Papaver constitutive AY2688942.6-2.9, methyltransferase coclaurine somniferum, overexpression,AY610507 3.6, 3.8, Norlaudanosoline → Thalicitum synthetic D29811 3.10,3.12 3′hydroxycoclaurine flavum, Coptis regulation ACO90225.1 EC2.1.1.128 japonica, BAM37634.1 Papaver bracteatum, Eschscholziacalifornica Coclaurine-N- CNMT Coclaurine → N- P. somniferum,constitutive AY217336 2.7-2.9 methyltransferase methylcoclaurine T.flavum, overexpression, AY610508 3.7, 3.8, 3′hydroxycoclaurine → 3′-Coptis japonica synthetic AB061863 3.11, 3.12 hydroxy-N-methylcoclaurineregulation EC 2.1.1.140 Cytochrome CYP80B1 N-methylcoclaurine → 3′- P.somniferum, constitutive AAF61400.1 2.8, 2.9 P450 80B1hydroxy-N-methylcoclaurine E. californica, overexpression, AAC39453.1 T.flavum synthetic AAU20767.1 regulation NADPH: ATR1, NADPH + H⁺ + nArabidopsis constitutive AAC05021.1 2.8, 2.9 hemoprotein CPR oxidizedhemoprotein → thaliana, overexpression, CAB58576.1 oxidoreductaseNADP⁺ + n reduced P. somniferum synthetic CAB58575.1 also knownhemoprotein regulation as cytochrome P450 reductase 4′-O- 4′OMT3′-hydroxy-N- P. somniferum, constitutive AY217333, 2.9,methyltransferase methylcoclaurine → T. flavum, overexpression, AY2173343.9-3.12 reticuline Coptis japonica, synthetic AY610510 EC 2.1.1.116 E.californica regulation D29812 BAM37633.1

TABLE 2 Comparison of impurities that may be present in concentrate ofpoppy straw (or opium) and clarified yeast culture medium. Concentrateof Clarified Yeast Impurities: Poppy Straw Culture Medium InorganicSodium ✓ ✓ Magnesium ✓ ✓ Silicon ✓

(not in culture medium) Phosphorus ✓ ✓ Sulfur ✓ ✓ Chloride ✓ ✓ Potassium✓ ✓ Calcium ✓ ✓ Copper ✓ ✓ Zinc ✓ ✓ Molybdenum ✓ ✓ (sodium molybdate inmedium) Iron ✓ ✓ Manganese ✓ ✓ Ammonium ✓ ✓ Boron ✓ ✓ OrganicPolysaccharides ✓

(starch, cellulose, (yeast fed xylan) simple sugars) Lignin(p-cournaryl, ✓

coniferyl, sinapyl alcohols) Pigments (chlorophyll, ✓

anthocyanins, carotenoids) Flavonoids ✓

Phenanthreoids ✓

Latex, gum, and wax ✓

Rubisco ✓

Meconic acid ✓

Pseudomorphine ✓

Narceine ✓

Thebaol ✓

Other Pesticides, Fungicides, ✓

Herbicides Pollen ✓

TABLE 3 Distinct groups of molecules present in clarified-yeast culturemedium (CYCM). Unlike concentrate of poppy straw (CPS) or opium, yeasthost strains may be engineered to produce molecules of a predeterminedclass of alkaloids (i.e., only one biosynthesis pathway per strain) suchthat other classes of alkaloids are not present. Therefore, the CYCM maycontain molecules within a single biosynthesis pathway including asubset of molecules spanning one or two columns, whereas the CPS maycontain a subset of molecules across many columns. Protoberberine and1-Benzylisoquinoline Phthalideisoquinoline Morphinan Isopavine AporphineBisBIA Tetrahydropapaverine Scoulerine Salutaridine Pavine MagnoflorineDauricine Dihydropapaverine Chelanthifoline Salutaridinol CaryachineCorytuberine Berbamunine Papaverine Stylopine Salutaridine-7-O-acetateBisnorargemonine Apomorphine Ligensinine Cis-N-methylstytopine ThebaineIsonoraremonine Boldine Fangchinoline Protopine Codeinone TetrandrineDihydrosanguinarine Oripavine Curine Sanguinerine MorphinoneCepharanthine Tetrahydrocolumbamine Neopinone Berbamine Canadine NeopineN-methylcanadine Codeine Noscapine Morphine Berberine NeomorphineNarcotoline Hydrocodone Narcotinehemiacetal OxycodoneNarcotolinehemiacetal 14-hydroxycodeinone 14-hydrocycodeineDihydromorphine Dihydrocodeine Oxymorphone Hydromorphone

Notwithstanding the appended clauses, the disclosure is also defined bythe following clauses:

1. A method for forming a product stream having a benzylisoquinolinealkaloid product, the method comprising:

(a) providing engineered yeast cells and a feedstock including nutrientsand water to a batch reactor, which engineered yeast cells have at leastone modification that results in overproduction of tyrosine with respectto a non-engineered yeast cell, wherein the at least one modification isselected from the group consisting of: a feedback inhibition alleviatingmutation in a biosynthetic enzyme gene and an inactivating mutation inan enzyme;

(b) in the batch reactor, subjecting the engineered yeast cells tofermentation by incubating the engineered yeast cells for a time periodof at least about 5 minutes to produce a solution comprising thebenzylisoquinoline alkaloid product and cellular material; and

(c) using at least one separation unit to separate thebenzylisoquinoline alkaloid product from the cellular material toprovide the product stream comprising the benzylisoquinoline alkaloidproduct.

2. The method of clause 1, wherein the engineered yeast cell comprisesone or more feedback inhibition alleviating mutations in one or morebiosynthetic enzyme genes that encode3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase.

3. The method of clause 2, wherein the one or more feedback inhibitionalleviating mutations are present in the3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase gene.

4. The method of clause 1, wherein the engineered yeast cell comprisesone or more feedback inhibition alleviating mutations in one or morebiosynthetic enzyme genes that encode chorismate mutase.

5. The method of clause 4, wherein the one or more feedback inhibitionalleviating mutations are present in the chorismate mutase gene.

6. The method of clause 1, wherein the engineered yeast cell furthercomprises at least one transcriptional modulation modification in abiosynthetic enzyme gene.

7. The method of clause 1, wherein at least one process parameter of thebatch reactor is modifiable to alter a resultant benzylisoquinolinealkaloid product composition.

8. The method of clause 7, wherein the at least one process parameterthat is modifiable comprises at least one of dissolved oxygen, pH,stirring speed, aeration rate, and cell density.

9. The method of clause 1, wherein the benzylisoquinoline alkaloidproduct comprises a benzylisoquinoline alkaloid precursor.

10. The method of clause 9, wherein the benzylisoquinoline alkaloidprecursor is selected from the group of norcoclaurine, norlaudanosoline,tyrosine, tyramine, 4-hydroxyphenylacetaldehyde, 4-hydroxyphenylpyruvicacid, L-3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetaldehyde, anddopamine.11. The method of clause 1, wherein the benzylisoquinoline alkaloidproduct comprises a benzylisoquinoline alkaloid.12. The method of clause 11, wherein the benzylisoquinoline alkaloid hasa structural class that is selected from the group ofbenzylisoquinolines, protoberberines, protopines, benzophenanthridines,promorphinans, morphinans, secoberberines, phthalideisoquinolines,aporphines, and bisbenzylisoquinolines.13. The method of clause 12, wherein the benzylisoquinoline alkaloid isa benzylisoquinoline that is selected from the group of coclaurine,3′-hydroxycoclaurine, 4′-O-methylnorlaudanosoline,4′-O-methyl-laudanosoline, N-methylnorcoclaurine, laudanosoline,N-methylcoclaurine, 3′-hydroxy-N-methylcoclaurine, reticuline,norreticuline, papaverine, laudanine, laudanosine, tetrahydropapaverine,1,2-dihydropapaverine, and orientaline.14. The method of clause 12, wherein the benzylisoquinoline alkaloid isa protoberberine that is selected from the group of scoulerine,cheilanthifoline, stylopine, nandinine, jatrorrhizine, stepholidine,discretamine, cis-N-methylstylopine, tetrahydrocolumbamine, palmatine,tetrahydropalmatine, columbamine, canadine, N-methylcanadine,1-hydroxycanadine, berberine, N-methyl-ophiocarpine,1,13-dihydroxy-N-methylcanadine, and1-hydroxy-10-O-acetyl-N-methylcanadine.15. The method of clause 12, wherein the benzylisoquinoline alkaloid isa protopine that is selected from the group of protopine,6-hydroxyprotopine, allocryptopine, cryptopine, muramine, andthalictricine.16. The method of clause 12, wherein the benzylisoquinoline alkaloid isa benzophenanthridine that is selected from the group ofdihydrosanguinarine, sanguinarine, dihydrocheilirubine, cheilirubine,dihydromarcapine, marcapine, and chelerythrine.17. The method of clause 12, wherein the benzylisoquinoline alkaloid isa promorphinan that is selected from the group of salutaridine,salutaridinol, and salutaridinol-7-O-acetate.18. The method of clause 12, wherein the benzylisoquinoline alkaloid isa morphinan that is selected from the group of thebaine, codeinone,codeine, morphine, morphinone, oripavine, neopinone, neopine,neomorphine, hydrocodone, dihydrocodeine, 14-hydroxycodeinone,oxycodone, 14-hydroxycodeine, morphinone, hydromorphone,dihydromorphine, dihydroetorphine, ethylmorphine, etorphine, metopon,buprenorphine, pholcodine, heterocodeine, and oxymorphone.19. The method of clause 12, wherein the benzylisoquinoline alkaloid isa secoberberine that is selected from the group of4′-O-desmethylmacrantaldehyde, 4′-O-desmethylpapaveroxine,4′-O-desmethyl-3-O-acetylpapaveroxine, and 3-O-aceteylpapaveroxine.20. The method of clause 12, wherein the benzylisoquinoline alkaloid isa phthalideisoquinoline that is selected from the group ofnarcotolinehemiacetal, narcotinehemiacetal, narcotoline, and noscapine.21. The method of clause 12, wherein the benzylisoquinoline alkaloid isan aporphine that is selected from the group of magnoflorine,corytuberine, apomorphine, boldine, isoboldine, isothebaine,isocorytuberine, and glaufine.22. The method of clause 12, wherein the benzylisoquinoline alkaloid isa bisbenzylisoquinoline that is selected from the group of berbamunine,guattgaumerine, dauricine, and liensinine.23. A method for forming a product stream having a benzylisoquinolinealkaloid product, the method comprising:

(a) providing engineered yeast cells and a feedstock including nutrientsand water to a reactor;

(b) in the reactor, subjecting the engineered yeast cells tofermentation by incubating the engineered yeast cells for a time periodof at least about 5 minutes to produce a solution comprising cellularmaterial and the benzylisoquinoline alkaloid product, wherein thesolution comprises not more than one class of molecule selected from thegroup of protoberberine, morphinan, isopavine, aporphine, andbisbenzylisoquinoline; and

(c) using at least one separation unit to separate thebenzylisoquinoline alkaloid product from the cellular material toprovide the product stream comprising the benzylisoquinoline alkaloidproduct.

24. The method of clause 23, wherein the engineered yeast cells have atleast one modification that results in overproduction of tyrosine withrespect to a non-engineered yeast cell, wherein the at least onemodification is selected from the group consisting of: a feedbackinhibition alleviating mutation in a biosynthetic enzyme gene and aninactivating mutation in an enzyme.25. The method of clause 24, wherein the engineered yeast cell furthercomprises at least one transcriptional modulation modification in abiosynthetic enzyme gene.26. The method of clause 23, wherein the product stream does not containmore than 5 ppm of a molecule selected from the group of lignin,pigments, flavonoids, phenanthreoids, latex, rubisco, meconic acid,pseudomorphine, narceine, thebaol, and pollen.27. The method of clause 26, wherein the product stream does not containmore than 5 ppm of meconic acid.28. The method of clause 23, wherein the product stream does not containa detectable amount of a substance selected from the group consisting ofpesticides, fungicides, or herbicides.29. The method of clause 23, wherein the benzylisoquinoline alkaloidproduct is recovered from the product stream through liquid-liquidextraction.30. The method of clause 29, wherein the benzylisoquinoline alkaloidproduct is recovered immediately after a fermentation process has beencompleted.31. The method of any of the previous clauses, wherein the engineeredyeast cell comprises two or more heterologous coding sequences, whereinthe two or more heterologous coding sequences encode at least a firstenzyme and a second enzyme that are involved in a metabolic pathway thatconverts the tyrosine into the benzylisoquinoline alkaloid product,wherein the first enzyme and second enzyme are operably connected alongthe metabolic pathway.32. The method of any of the previous clauses, wherein the engineeredyeast cell comprises three heterologous coding sequences, wherein thethree heterologous coding sequences encode a first enzyme, secondenzyme, and third enzyme that are involved in a metabolic pathway thatconverts the tyrosine into the benzylisoquinoline alkaloid product,wherein the first enzyme, second enzyme, and third enzyme are operablyconnected along the metabolic pathway.33. An engineered yeast cell that produces a benzylisoquinoline alkaloidproduct, the engineered yeast cell having at least one modification thatresults in overproduction of tyrosine with respect to a non-engineeredyeast cell, wherein the at least one modification is selected from thegroup consisting of: a feedback inhibition alleviating mutation in abiosynthetic enzyme gene and an inactivating mutation in an enzyme,

wherein the engineered yeast cell comprises at least one heterologouscoding sequence encoding at least one enzyme that is selected from thegroup of tyrosine hydroxylase, L-DOPA decarboxylase, and norcoclaurinesynthase.

34. The engineered yeast cell of clause 33, wherein the engineered yeastcell further comprises at least one heterologous coding sequenceencoding at least one enzyme that is selected from the group ofnorcoclaurine 6-O-methyltransferase, coclaurine-N-methyltransferase,cytochrome P450 8061, cytochrome P450 reductase, and4′-O-methyltransferase.35. The engineered yeast cell of clause 34, wherein thebenzylisoquinoline alkaloid product comprises at least one ofcoclaurine, N-methylcoclaurine, 3′hydroxy-N-methyl-coclaurine,3′hydroxycoclaurine, laudanosoline, 4′-O-methyllaudanosoline,norreticuline, and reticuline.36. A method for forming a product stream having a benzylisoquinolinealkaloid product, the method comprising:

(a) providing engineered non-plant cells and a feedstock includingnutrients and water to a batch reactor, which engineered non-plant cellshave at least one modification that results in overproduction oftyrosine with respect to a non-engineered non-plant cell, wherein the atleast one modification is selected from the group consisting of: afeedback inhibition alleviating mutation in a biosynthetic enzyme geneand an inactivating mutation in an enzyme;

(b) in the batch reactor, subjecting the engineered non-plant cells tofermentation by incubating the engineered non-plant cells for a timeperiod of at least about 5 minutes to produce a solution comprising thebenzylisoquinoline alkaloid product and cellular material; and

(c) using at least one separation unit to separate thebenzylisoquinoline alkaloid product from the cellular material toprovide the product stream comprising the benzylisoquinoline alkaloidproduct.

37. The method of clause 36, wherein the engineered non-plant cellcomprises one or more feedback inhibition alleviating mutations in oneor more biosynthetic enzyme genes that encode3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase.

38. The method of clause 37, wherein the one or more feedback inhibitionalleviating mutations are present in the3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase gene.

39. The method of clause 36, wherein the engineered non-plant cellcomprises one or more feedback inhibition alleviating mutations in oneor more biosynthetic enzyme genes that encode chorismate mutase.

40. The method of clause 39, wherein the one or more feedback inhibitionalleviating mutations are present in the chorismate mutase gene.

41. The method of clause 36, wherein the engineered non-plant cellfurther comprises at least one transcriptional modulation modificationin a biosynthetic enzyme gene.

42. The method of clause 36, wherein at least one process parameter ofthe batch reactor is modifiable to alter a resultant benzylisoquinolinealkaloid product composition.

43. The method of clause 42, wherein the at least one process parameterthat is modifiable comprises at least one of dissolved oxygen, pH,stirring speed, aeration rate, and cell density.

44. The method of clause 36, wherein the benzylisoquinoline alkaloidproduct comprises a benzylisoquinoline alkaloid precursor.

45. The method of clause 44, wherein the benzylisoquinoline alkaloidprecursor is selected from the group of norcoclaurine, norlaudanosoline,tyrosine, tyramine, 4-hydroxyphenylacetaldehyde, 4-hydroxyphenylpyruvicacid, L-3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetaldehyde, anddopamine.46. The method of clause 36, wherein the benzylisoquinoline alkaloidproduct comprises a benzylisoquinoline alkaloid.47. The method of clause 46, wherein the benzylisoquinoline alkaloid hasa structural class that is selected from the group ofbenzylisoquinolines, protoberberines, protopines, benzophenanthridines,promorphinans, morphinans, secoberberines, phthalideisoquinolines,aporphines, and bisbenzylisoquinolines.48. The method of clause 47, wherein the benzylisoquinoline alkaloid isa benzylisoquinoline that is selected from the group of coclaurine,3′-hydroxycoclaurine, 4′-O-methylnorlaudanosoline,4′-O-methyl-laudanosoline, N-methylnorcoclaurine, laudanosoline,N-methylcoclaurine, 3′-hydroxy-N-methylcoclaurine, reticuline,norreticuline, papaverine, laudanine, laudanosine, tetrahydropapaverine,1,2-dihydropapaverine, and orientaline.49. The method of clause 47, wherein the benzylisoquinoline alkaloid isa protoberberine that is selected from the group of scoulerine,cheilanthifoline, stylopine, nandinine, jatrorrhizine, stepholidine,discretamine, cis-N-methylstylopine, tetrahydrocolumbamine, palmatine,tetrahydropalmatine, columbamine, canadine, N-methylcanadine,1-hydroxycanadine, berberine, N-methyl-ophiocarpine,1,13-dihydroxy-N-methylcanadine, and1-hydroxy-10-O-acetyl-N-methylcanadine.50. The method of clause 47, wherein the benzylisoquinoline alkaloid isa protopine that is selected from the group of protopine,6-hydroxyprotopine, allocryptopine, cryptopine, muramine, andthalictricine.51. The method of clause 47, wherein the benzylisoquinoline alkaloid isa benzophenanthridine that is selected from the group ofdihydrosanguinarine, sanguinarine, dihydrocheilirubine, cheilirubine,dihydromarcapine, marcapine, and chelerythrine.52. The method of clause 47, wherein the benzylisoquinoline alkaloid isa promorphinan that is selected from the group of salutaridine,salutaridinol, and salutaridinol-7-O-acetate.53. The method of clause 47, wherein the benzylisoquinoline alkaloid isa morphinan that is selected from the group of thebaine, codeinone,codeine, morphine, morphinone, oripavine, neopinone, neopine,neomorphine, hydrocodone, dihydrocodeine, 14-hydroxycodeinone,oxycodone, 14-hydroxycodeine, morphinone, hydromorphone,dihydromorphine, dihydroetorphine, ethylmorphine, etorphine, metopon,buprenorphine, pholcodine, heterocodeine, and oxymorphone.54. The method of clause 47, wherein the benzylisoquinoline alkaloid isa secoberberine that is selected from the group of4′-O-desmethylmacrantaldehyde, 4′-O-desmethylpapaveroxine,4′-O-desmethyl-3-O-acetylpapaveroxine, and 3-O-aceteylpapaveroxine.55. The method of clause 47, wherein the benzylisoquinoline alkaloid isa phthalideisoquinoline that is selected from the group ofnarcotolinehemiacetal, narcotinehemiacetal, narcotoline, and noscapine.56. The method of clause 47, wherein the benzylisoquinoline alkaloid isan aporphine that is selected from the group of magnoflorine,corytuberine, apomorphine, boldine, isoboldine, isothebaine,isocorytuberine, and glaufine.57. The method of clause 47, wherein the benzylisoquinoline alkaloid isa bisbenzylisoquinoline that is selected from the group of berbamunine,guattgaumerine, dauricine, and liensinine.58. A method for forming a product stream having a benzylisoquinolinealkaloid product, the method comprising:

(a) providing engineered non-plant cells and a feedstock includingnutrients and water to a reactor;

(b) in the reactor, subjecting the engineered non-plant cells tofermentation by incubating the engineered non-plant cells for a timeperiod of at least about 5 minutes to produce a solution comprisingcellular material and the benzylisoquinoline alkaloid product, whereinthe solution comprises not more than one class of molecule selected fromthe group of protoberberine, morphinan, isopavine, aporphine, andbisbenzylisoquinoline; and

(c) using at least one separation unit to separate thebenzylisoquinoline alkaloid product from the cellular material toprovide the product stream comprising the benzylisoquinoline alkaloidproduct.

59. The method of clause 58, wherein the engineered non-plant cells haveat least one modification that results in overproduction of tyrosinewith respect to a non-engineered non-plant cell, wherein the at leastone modification is selected from the group consisting of: a feedbackinhibition alleviating mutation in a biosynthetic enzyme gene and aninactivating mutation in an enzyme.60. The method of clause 59, wherein the engineered non-plant cellfurther comprises at least one transcriptional modulation modificationin a biosynthetic enzyme gene.61. The method of clause 58, wherein the product stream does not containmore than 5 ppm of a molecule selected from the group of lignin,pigments, flavonoids, phenanthreoids, latex, rubisco, meconic acid,pseudomorphine, narceine, thebaol, and pollen.62. The method of clause 61, wherein the product stream does not containmore than 5 ppm of meconic acid.63. The method of clause 58, wherein the product stream does not containa detectable amount of a substance selected from the group consisting ofpesticides, fungicides, or herbicides.64. The method of clause 58, wherein the benzylisoquinoline alkaloidproduct is recovered from the product stream through liquid-liquidextraction.65. The method of clause 64, wherein the benzylisoquinoline alkaloidproduct is recovered immediately after a fermentation process has beencompleted.66. The method of any of the previous clauses, wherein the engineerednon-plant cell comprises two or more heterologous coding sequences,wherein the two or more heterologous coding sequences encode at least afirst enzyme and a second enzyme that are involved in a metabolicpathway that converts the tyrosine into the benzylisoquinoline alkaloidproduct, wherein the first enzyme and second enzyme are operablyconnected along the metabolic pathway.67. The method of any of the previous clauses, wherein the engineerednon-plant cell comprises three heterologous coding sequences, whereinthe three heterologous coding sequences encode a first enzyme, secondenzyme, and third enzyme that are involved in a metabolic pathway thatconverts the tyrosine into the benzylisoquinoline alkaloid product,wherein the first enzyme, second enzyme, and third enzyme are operablyconnected along the metabolic pathway.68. An engineered non-plant cell that produces a benzylisoquinolinealkaloid product, the engineered non-plant cell having at least onemodification that results in overproduction of tyrosine with respect toa non-engineered non-plant cell, wherein the at least one modificationis selected from the group consisting of: a feedback inhibitionalleviating mutation in a biosynthetic enzyme gene and an inactivatingmutation in an enzyme,

wherein the engineered non-plant cell comprises at least oneheterologous coding sequence encoding at least one enzyme that isselected from the group of tyrosine hydroxylase, L-DOPA decarboxylase,and norcoclaurine synthase.

69. The engineered non-plant cell of clause 68, wherein the engineerednon-plant cell further comprises at least one heterologous codingsequence encoding at least one enzyme that is selected from the group ofnorcoclaurine 6-O-methyltransferase, coclaurine-N-methyltransferase,cytochrome P450 8061, cytochrome P450 reductase, and4′-O-methyltransferase.70. The engineered non-plant cell of clause 69, wherein thebenzylisoquinoline alkaloid product comprises at least one ofcoclaurine, N-methylcoclaurine, 3′hydroxy-N-methyl-coclaurine,3′hydroxycoclaurine, laudanosoline, 4′-O-methyllaudanosoline,norreticuline, and reticuline.71. The engineered non-plant cell of any of the previous clauses,wherein the engineered non-plant cell comprises two or more heterologouscoding sequences, wherein the two or more heterologous coding sequencesencode at least a first enzyme and a second enzyme that are involved ina metabolic pathway that converts the tyrosine into thebenzylisoquinoline alkaloid product, wherein the first enzyme and secondenzyme are operably connected along the metabolic pathway.72. The engineered non-plant cell of any of the previous clauses,wherein the engineered non-plant cell comprises three heterologouscoding sequences, wherein the three heterologous coding sequences encodea first enzyme, second enzyme, and third enzyme that are involved in ametabolic pathway that converts the tyrosine into the benzylisoquinolinealkaloid product, wherein the first enzyme, second enzyme, and thirdenzyme are operably connected along the metabolic pathway.73. A compound that comprises:

a benzylisoquinoline alkaloid product that is characterized as beingpart of at most two classes selected from the group consisting of1-benzylisoquinoline, protoberberine, morphinan, isopavine, aporphine,and bisbenzylisoquinoline,

wherein remaining components of the compound do not contain a detectableamount of a molecule of a non-selected class from the group of1-benzylisoquinoline, protoberberine, morphinan, isopavine, aporphine,and bisbenzylisoquinoline.

74. The compound of clause 73, wherein the benzylisoquinoline alkaloidproduct is characterized as having a detectable amount of at most oneclass selected from the group consisting of 1-benzylisoquinoline,protoberberine, morphinan, isopavine, aporphine, andbisbenzylisoquinoline.75. The compound of clause 73, wherein the benzylisoquinoline alkaloidproduct is characterized as being part of the 1-benzylisoquinoline classand also being part of at most one class selected from the groupconsisting of protoberberine, morphinan, isopavine, aporphine, andbisbenzylisoquinoline.76. A therapeutic agent that comprises:

a benzylisoquinoline alkaloid product,

wherein the therapeutic agent does not contain a detectable amount of animpurity selected from the group consisting of codeine-O(6)-methylether, 8,14-dihydroxy-7,8-dihydrocodeinone, and tetrahydrothebaine.

77. The method of any of clauses 23-32, wherein the solution comprisesno detectable amount of molecules selected from the group ofprotoberberine, morphinan, isopavine, aporphine, andbisbenzylisoquinoline, and wherein the solution comprises moleculeswithin a class of phthalideisoquinolines.78. The method of any of clauses 23-32, wherein the solution comprisesmolecules within the class of protoberberine, and wherein the solutionfurther comprises molecules within a class of phthalideisoquinolines.79. The method of any of clauses 58-67, wherein the solution comprisesno detectable amount of molecules selected from the group ofprotoberberine, morphinan, isopavine, aporphine, andbisbenzylisoquinoline, and wherein the solution comprises moleculeswithin a class of phthalideisoquinolines.80. The method of any of clauses 58-67, wherein the solution comprisesmolecules within the class of protoberberine, and wherein the solutionfurther comprises molecules within a class of phthalideisoquinolines.81. The compound of any of clauses 73-75, wherein the benzylisoquinolinealkaloid product is not classified as being part of a class selectedfrom the group consisting of 1-benzylisoquinoline, protoberberine,morphinan, isopavine, aporphine, and bisbenzylisoquinoline, and whereinthe benzylisoquinoline alkaloid product is classified as being part of aphthalideisoquinoline alkaloid class.82. The method of any of clauses 73-75, wherein the benzylisoquinolinealkaloid product is classified as being part of the protoberberine classand the phthalideisoquinoline class.83. The method of any of clauses 73-75, wherein the benzylisoquinolinealkaloid product is classified as being part of the 1-benzylisoquinolineclass, the protoberberine class, and the phthalideisoquinoline class.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

What is claimed is:
 1. A method of producing a pharmaceutical opioidformulation, the method comprising: feeding a plurality of engineerednon-plant cells with a starting material in a cellular growth medium;producing a pharmaceutical opioid compound, or a precursor thereof, fromthe plurality of engineered non-plant cells to create a mixturecomprising the plurality of engineered non-plant cells, the cellulargrowth medium, and the pharmaceutical opioid compound or a precursorthereof; processing the pharmaceutical opioid compound or precursorthereof, wherein the processing comprises: separating out engineerednon-plant cells using at least one process selected from the groupconsisting of sedimentation, filtration, and centrifugation; andproducing the pharmaceutical opioid formulation that comprises thepharmaceutical opioid compound, wherein the mixture does not contain adetectable amount of a compound selected from the group consisting ofsilicon, polysaccharides, lignin, pigments, flavonoids, phenanthreoids,latex, gum, wax, rubisco, meconic acid, pseudomorphine, narceine,thebaol, pesticides, fungicides, herbicides, and pollen.
 2. The methodof claim 1, wherein the pharmaceutical opioid formulation comprises anactive ingredient of the pharmaceutical opioid compound.
 3. The methodof claim 2, wherein the active ingredient comprises the pharmaceuticalopioid compound with 98% or greater purity.
 4. The method of claim 1,wherein feeding comprises fermenting the plurality of engineerednon-plant cells with a starting material in the cellular growth medium.5. The method of claim 1, wherein feeding occurs within a cell-culturingdevice that is selected from the group consisting of a batch reactor anda continuous-flow reactor.
 6. The method of claim 5, wherein thecell-culturing device comprises a fed-batch reactor.
 7. The method ofclaim 1, wherein the pharmaceutical opioid compound is an impuritywithin an active ingredient within the pharmaceutical opioidformulation.
 8. The method of claim 1, wherein the cellular growthmedium comprises an organic molecule as a starting material.
 9. Themethod of claim 8, wherein the organic molecule comprises amonosaccharide, an oligosaccharide, a polysaccharide, an unpurifiedrenewable feedstock, a one-carbon substrate or a two-carbon substrate,or a combination thereof.
 10. The method of claim 1, wherein theplurality of engineered non-plant cells comprises a plurality ofmammalian cells, a plurality of insect cells, a plurality of bacteriacells or a plurality of fungal cells, or a combination thereof.
 11. Themethod of claim 10, wherein the plurality of engineered non-plant cellscomprises the plurality of fungal cells.
 12. The method of claim 11,wherein the plurality of engineered non-plant cells comprises theplurality of yeast cells.
 13. The method of claim 10, wherein theplurality of engineered non-plant cells comprises the plurality ofbacterial cells.
 14. The method of claim 1, wherein the pharmaceuticalopioid compound or precursor thereof comprises an alkaloid from the1-benzylisoquinoline, promorphinan, or morphinan class.
 15. The methodof claim 14, wherein the pharmaceutical opioid compound comprises analkaloid of the morphinan class.
 16. The method of claim 15, wherein thealkaloid comprises codeine, morphine, hydrocodone, hydromorphone,oxycodone, dihydrocodeine or oxymorphone.
 17. The method of claim 15,wherein the processing further comprises performing at least one processselected from the group consisting of: a chemical reaction, a chemicalseparation, and a phase separation.
 18. The method of claim 17, whereinthe further processing comprises performing a chemical reaction thatcomprises a non-enzymatic chemical reaction.
 19. The method of claim 17,wherein the further processing comprises a chemical separation that isselected from the group consisting of: adsorbing, performingchromatography, emulsifying, extracting, and a combination thereof. 20.The method of claim 17, wherein the further processing comprises a phaseseparation that is selected from the group consisting of filtering,chelating, centrifuging, crystallizing, and a combination thereof. 21.The method of claim 19, wherein the performing chromatography comprisesa chromatography selected from the group consisting of: high-pressureliquid chromatography, size exclusion chromatography, and normal-phasechromatography.
 22. The method of claim 19, wherein the extractingcomprises liquid extraction or pH-based purification.
 23. The method ofclaim 1, further comprising: administering the pharmaceutical opioidformulation to a subject.
 24. The method of claim 23, wherein theadministering is for a therapeutic purpose.
 25. The method of claim 2,wherein the pharmaceutical opioid formulation comprises a second activeingredient.
 26. The method of claim 2, wherein the pharmaceutical opioidformulation comprises more than two active ingredients.
 27. The methodof claim 1, wherein the pharmaceutical opioid compound, or precursorthereof, is excreted from the plurality of engineered non-plant cells.28. The method of claim 1, wherein the pharmaceutical opioid compound,or precursor thereof, is diffused from the plurality of engineerednon-plant cells.
 29. The method of claim 1, wherein the pharmaceuticalopioid compound, or precursor thereof, is provided to the cellulargrowth medium from the plurality of engineered non-plant cells throughtransport.